Bone Matrix Compositions and Methods

ABSTRACT

An osteoinductive composition, corresponding osteoimplants, and methods for making the osteoinductive composition are disclosed. The osteoinductive composition comprises osteoinductive factors, such as may be extracted from demineralized bone, and a carrier. The osteoinductive composition is prepared by providing demineralized bone, extracting osteoinductive factors from the demineralized bone, and adding the extracted osteoinductive factors to a carrier. Further additives such as bioactive agents may be added to the osteoinductive composition. The carrier and osteoinductive factors may form an osteogenic osteoimplant. The osteoimplant, when implanted in a mammalian body, can induce at the locus of the implant the full developmental cascade of endochondral bone formation including vascularization, mineralization, and bone marrow differentiation. Also, in some embodiments, the osteoinductive composition can be used as a delivery device to administer bioactive agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/732,675, filed on Nov. 1, 2005, the contents of which areincorporated in its entirety by reference herein.

BACKGROUND

Introduction

Mammalian bone tissue is known to contain one or more proteinaceousmaterials, presumably active during growth and natural bone healing,that can induce a developmental cascade of cellular events resulting inendochondral bone formation. The active factors are variously beenreferred to in the literature as bone morphogenetic or morphogenicproteins (BMPs), bone inductive proteins, bone growth or growth factors,osteogenic proteins, or osteoinductive proteins. These active factorsare collectively referred to herein as osteoinductive factors.

It is well known that bone contains these osteoinductive factors. Theseosteoinductive factors are present within the compound structure ofcortical bone and are present at very low concentrations, e.g., 0.003%.Osteoinductive factors direct the differentiation of pluripotentialmesenchymal cells into osteoprogenitor cells that form osteoblasts.Based upon the work of Marshall Urist as shown in U.S. Pat. No.4,294,753, issued Oct. 13, 1981, proper demineralization of corticalbone exposes the osteoinductive factors, rendering it osteoinductive, asdiscussed more fully below.

Overview of Bone Grafts

The rapid and effective repair of bone defects caused by injury,disease, wounds, or surgery has long been a goal of orthopaedic surgery.Toward this end, a number of compositions and materials have been usedor proposed for use in the repair of bone defects. The biological,physical, and mechanical properties of the compositions and materialsare among the major factors influencing their suitability andperformance in various orthopaedic applications.

Autologous cancellous bone (“ACB”) long has been considered the goldstandard for bone grafts. ACB is osteoinductive and nonimmunogenic, and,by definition, it has all of the appropriate structural and functionalcharacteristics appropriate for the particular recipient. Unfortunately,ACB is only available in a limited number of circumstances. Someindividuals lack ACB of appropriate dimensions and quality fortransplantation, and donor site pain and morbidity can pose seriousproblems for patients and their physicians.

Much effort has been invested in the identification and development ofalternative bone graft materials. Urist has published seminal articleson the theory of bone induction and a method for decalcifying bone,i.e., making demineralized bone matrix (DBM). Urist M. R., BoneFormation by Autoinduction, Science 1965; 150(698):893-9; Urist M. R. etal., The Bone Induction Principle, Clin. Orthop. Rel. Res. 53:243-283,1967. As mentioned above, DBM is an osteoinductive material, in that itinduces bone growth when implanted in an ectopic site of a rodent, owingto the osteoinductive factors contained within the DBM. Honsawek et al.(2000). It is now known that there are numerous osteoinductive factors,e.g., BMP 1-15, which are part of the transforming growth factor-beta(TGF-beta) superfamily (Kawabata et al., 2000). BMP-2 has become themost important and widely studied of the BMP family of proteins. Thereare also other proteins present in DBM that are not osteoinductive alonebut still contribute to bone growth, including fibroblast growthfactor-2 (FGF-2), insulin-like growth factor-I and -II (IGF-I andIGF-II), platelet derived growth factor (PDGF), and transforming growthfactor-beta 1 (TGF-beta.1) (Hauschka, et al. 1986; Canalis, et al, 1988;Mohan et al. 1996).

DBM implants have been reported to be particularly useful (see, forexample, U.S. Pat. Nos. 4,394,370, 4,440,750, 4,485,097, 4,678,470, and4,743,259; Mulliken et al., Calcif Tissue Int. 33:71, 1981; Neigel etal., Opthal. Plast. Reconstr. Surg. 12:108, 1996; Whiteman et al., J.Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin. Orthop. 293:360, 1993,each of which is incorporated herein by reference). DBM typically isderived from cadavers. The bone is removed aseptically and treated tokill any infectious agents. The bone is particulated by milling orgrinding, and then the mineral component is extracted by variousmethods, such as by soaking the bone in an acidic solution. Theremaining matrix is malleable and can be further processed and/or formedand shaped for implantation into a particular site in the recipient.Demineralized bone prepared in this manner contains a variety ofcomponents including proteins, glycoproteins, growth factors, andproteoglycans. Following implantation, the presence of DBM inducescellular recruitment to the site of injury. The recruited cells mayeventually differentiate into bone forming cells. Such recruitment ofcells leads to an increase in the rate of wound healing and, therefore,to faster recovery for the patient.

Some studies indicate that the osteoinductive capabilities ofdemineralized bone from higher order species in higher order species isrelatively low. One study compared the osteoinductivity of rat andcanine bone matrix, and of cortical and cancellous bone. Rat bone matrixconsistently induced new bone and high phosphatase levels when implantedectopically in rat. Canine matrix induced small amounts of bone andlower phosphatase levels when implanted in dog and in rat, with corticalmatrix being somewhat more inductive than cancellous matrix.Demineralized cancellous bone matrix from dog was the only materialtested not showing any osteoinductivity. Schwarz et al., Acta. Orthop.Scan. 60(6):693-695, 1989.

Similarly, another study determined that monkey bone matrix inducesectopic bone formation in the athymic rat but not in adult monkeys. Itwas concluded that adult monkey bone matrix contains bone inductiveproperties but that these properties are not sufficient to induce boneformation in adult monkey muscle sites. Aspenberg et al., J. of Orthop.Res. 9:20-25, 1991.

Yet another study evaluated bone and cementum regeneration followingguided tissue regeneration (GTR) in periodontal fenestration defects.Specifically, the adjunctive effect of allogenic, freeze-dried DBMimplant was evaluated and found to exhibit no discernible adjunctiveeffect to GTR in the defect model. The critical findings were 1) the DBMparticles remained embedded in dense connective tissue without evidenceof bone metabolic activity; and 2) limited and similar amounts of boneand cementum regeneration were observed for both GTR plus DBM and GTRdefects. Caplanis et al., J Periodontal 851-856, August, 1998.

Current DBM formulations have various drawbacks. First, while thecollagen-based matrix of DBM is relatively stable, the osteoinductivefactors within the DBM matrix are rapidly degraded. The osteogenicactivity of the DBM may be significantly degraded within 24 hours afterimplantation, and in some instances the osteogenic activity may beinactivated within 6 hours. Therefore, the osteoinductive factorsassociated with the DBM are only available to recruit cells to the siteof injury for a short time after transplantation. For much of thehealing process, which may take weeks to months, the implanted materialmay provide little or no assistance in recruiting cells. In addition tothe osteoinductive factors present within the DBM, the overall structureof the DBM implant is also believed to contribute to the bone healingcapabilities of the implant.

Extracting Proteins

The potential utility of osteoinductive factors has been recognizedwidely. It has been contemplated that the availability of osteoinductivefactors could revolutionize orthopedic medicine and certain types ofplastic surgery, dental, and various periodontal and craniofacialreconstructive procedures.

Urist's U.S. Pat. No. 4,294,753, herein incorporated by reference, wasthe first of his many patents on a process for extracting BMP from DBM.At the time of the Urist '753 patent, BMP was referred to generally.However, as mentioned above, now it is known that there are multipleforms of BMP. The Urist process became widely adopted, and thoughdifferent users may use different chemical agents from those disclosedin the basic Urist process, the basic layout of the steps of the processremains widely used today as one of the main methods of extracting BMPfrom DBM. See, e.g., U.S. Pub 2003/0065392 (2003); U.S. Pub 2002/0197297(2002). Urist reported that his basic process actually results ingenerally low yields of BMP per unit weight of DBM. Urist et al. (1982).

The observed properties of osteoinductive factors have induced anintense research effort in several laboratories directed to isolatingand identifying the pure factor or factors responsible for osteogenicactivity. A modified process for purification of osteogenic protein frommammalian bone is disclosed by Sampath et al. (1987) Proc. Natl. Acad.Sci. USA 84:7109-7113. Urist et al. (1983), Proc. Soc. Exp. Biol. Med.173:194-199, disclose a human osteogenic protein fraction which wasextracted from demineralized cortical bone by means of a calciumchloride-urea inorganic-organic solvent mixture, and retrieved bydifferential precipitation in guanidine-hydrochloride and preparativegel electrophoresis. The authors report that the protein fraction has anamino acid composition of an acidic polypeptide and a molecular weightin a range of 17-18 kDa. This material was said to be distinct from aprotein called “bone derived growth factor” disclosed by Canalis et al.(1980 Science 210:1021-1023) and by Farley et al. (1982) Biochem21:3508-3513.

Urist et al., (1984) Proc. Natl. Acad. Sci. USA 81:371-375, disclose abovine BMP extract having the properties of an acidic polypeptide and amolecular weight of approximately 18 kDa. The authors report that theprotein was present in a fraction separated by hydroxyapatitechromatography, and that it induced bone formation in mouse hindquartermuscle and bone regeneration in trephine defects in rat and dog skulls.Their method of obtaining the extract from bone results in ill-definedand impure preparations.

European Patent Application Serial No. 148,155, published Oct. 7, 1985,herein incorporated by reference, purports to disclose osteogenicproteins derived from bovine, porcine, and human origin. One of theproteins, designated by the inventors as a P3 protein having a molecularweight of 22-24 kDa, is said to have been purified to an essentiallyhomogeneous state. This material is reported to induce bone formationwhen implanted into animals.

International Application No. PCT/087/01537 (Int. Pub. No. WO88/00205)discloses an impure fraction from bovine bone with bone inductionqualities. The named applicants also disclose putative “bone inductivefactors” produced by recombinant DNA techniques. Four DNA sequences wereretrieved from human or bovine genomic or cDNA libraries and expressedin recombinant host cells. While the applicants stated that theexpressed proteins may be bone morphogenic proteins, bone induction wasnot demonstrated. This same group reported subsequently ((1988) Science242:1528-1534) that three of the four factors induce cartilageformation, and postulate that bone formation activity “is due to amixture of regulatory molecules” and that “bone formation is most likelycontrolled . . . by the interaction of these molecules.” Again, no boneinduction was attributed to the products of expression of the cDNAs. Seealso Urist et al., EPO 0,212,474, entitled “Bone Morphogenic Agents.”

Wang et al., (1988) Proc. Nat. Acad. Sci. USA 85: 9484-9488, disclosethe partial purification of a bovine bone morphogenetic protein fromguanidine extracts of demineralized bone having cartilage and boneformation activity as a basic protein corresponding to a molecularweight of 30 kDa determined from gel elution. Separation of the 30 kDafraction yielded proteins of 30, 18, and 16 kDa, which, upon separation,were inactive. In view of this result, the authors acknowledge that theexact identity of the active material had not been determined.

Wang et al., (1990) Proc. Nat. Acad. Sci. USA 87: 2220-2224, describethe expression and partial purification of one of the cDNA sequencesdescribed in PCT 87/01537. Consistent cartilage and/or bone formationwith their protein requires a minimum of 600 ng of 50% pure material.

International Application No. PCT/89/04458 (Int. Pub. No. WO90/003733)describes the purification and analysis of a family of osteogenicfactors called “P3 OF 31-34.”. The protein family contains at least fourproteins, which are characterized by peptide fragment sequences. Theimpure mixture P3 OF 31-34 is assayed for osteogenic activity. Theactivity of the individual proteins is neither assessed nor discussed.

Implanting Extracted Proteins

Successful implantation of the osteoinductive factors for endochondralbone formation requires association of the proteins with a suitablecarrier material capable of maintaining the proteins at an in vivo siteof application. The carrier should be biocompatible, in vivobiodegradable, and porous enough to allow cell infiltration. Insolublecollagen particles that remain after guanidine extraction anddelipidation of pulverized bone generally have been found effective inallogenic implants in some species. However, studies have shown thatwhile osteoinductive proteins are useful cross species, the collagenousbone matrix generally used for inducing endochondral bone formation isspecies-specific. Sampath and Reddi, (1983) Proc. Nat. Acad. Sci. USA80: 6591-6594. Demineralized, delipidated, extracted xenogenic bonematrix carriers implanted in vivo invariably fail to induceosteogenesis, presumably due to inhibitory or immunogenic components inthe bone matrix. Even the use of allogenic bone matrix in osteogenicdevices may not be sufficient for osteoinductive bone formation in manyspecies, as discussed above.

U.S. Pat. No. 4,563,350, herein incorporated by reference, discloses theuse of trypsinized bovine bone matrix as a xenogenic matrix to effectosteogenic activity when implanted with extracted, partially purifiedbone-inducing protein preparations. Bone formation is said to requirethe presence of at least 5%, and preferably at least 10%, non-fibrillarcollagen. The named inventors claim that removal of telopeptides thatare responsible in part for the immunogenicity of collagen preparationsis more suitable for xenogenic implants.

European Patent Application Serial No. 309,241, published Mar. 29, 1989,herein incorporated by reference, discloses a device for inducingendochondral bone formation comprising an osteogenic proteinpreparation, and a matrix carrier comprising 60-98% of either mineralcomponent or bone collagen powder and 2-40% atelopeptide hypoimmunogeniccollagen.

Deatherage et al., (1987) Collagen Rel. Res. 7: 2225-2231, purport todisclose an apparently xenogenic implantable device comprising a bovinebone matrix extract that has been minimally purified by a one-step ionexchange column and reconstituted with highly purified human Type-Iplacental collagen.

U.S. Pat. No. 3,394,370, herein incorporated by reference, describes amatrix of reconstituted collagen purportedly useful in xenogenicimplants. The collagen fibers are treated enzymatically to removepotentially immunogenic telopeptides (also the primary source ofinterfibril crosslinks), and are dissolved to remove associatednoncollagenenous components. The matrix is formulated by dispersing thereconstituted collagen in acetic acid to form a disordered matrix ofelementary collagen molecules that is then mixed with an osteogenicsubstance and lyophilized to form a “semi-rigid foam or sponge” that ispreferably crosslinked. The formulated matrix is not tested in vivo.

U.S. Pat. No. 4,172,128, herein incorporated by reference, describes amethod for degrading and regenerating bone-like material of reducedimmunogenicity, said to be useful cross-species. Demineralized boneparticles are treated with a swelling agent to dissolve any associatedmucopolysaccharides (glycosaminoglycans), and the collagen fiberssubsequently dissolved to form a homogenous colloidal solution. A gel ofreconstituted fibers then can be formed using physiologically inertmucopolysaccharides and an electrolyte to aid in fibril formation.

The use of pulverized exogenous bone growth material, e.g., derived fromdemineralized allogenic or xenogenic bone, in the surgical repair orreconstruction of defective or diseased bone is known. See, in thisregard, the disclosures of U.S. Pat. Nos. 4,394,370, 4,440,750,4,472,840, 4,485,097, 4,678,470, and 4,743,259; Bolander et al., “TheUse of Demineralized Bone Matrix in the Repair of Segmental Defects,”The Journal of Bone and Joint Surgery, Vol. 68-A, No. 8, pp. 1264-1273;Glowacki et al, “Demineralized Bone Implants,” Symposium on Horizons inPlastic Surgery, Vol. 12, No. 2; pp. 233-241 (1985); Gepstein et al.,“Bridging Large Defects in Bone by Demineralized Bone Matrix in the Formof a Powder,” The Journal of Bone and Joint Surgery, Vol. 69-A, No. 7,pp. 984-991 (1987); Mellonig, “Decalcified Freeze-Dried Bone Allograftas an Implant Material In Human Periodontal Defects,” The InternationalJournal of Periodontics and Restorative Dentistry, pp. 41-45 (June1984); Kaban et al., “Treatment of Jaw Defects with Demineralized BoneImplants,” Journal of Oral and Maxillofacial Surgery, pp. 623-626 (Jun.6, 1989); and Todescan et al., “A Small Animal Model for InvestigatingEndosseous Dental Implants: Effect of Graft Materials on Healing ofEndosseous, Porous-Surfaced Implants Placed in a Fresh ExtractionSocket,” The International Journal of Oral & Maxillofacial Implants Vol.2, No. 4, pp. 217-223 (1987), all herein incorporated by reference.

A variety of approaches have been explored in an attempt to recruitprogenitor cells or chondrocytes into an osteochondral or chondraldefect. For example, penetration of subchondral bone has been performedin order to access mesenchymal stem cells (MSCs) in the bone marrow,which have the potential to differentiate into cartilage and bone.Steadman, et al., “Microfracture: Surgical Technique and Rehabilitationto Treat Chondral Defects,” Clin. Orthop., 391 S:362-369 (2001). Inaddition, some factors in the body are believed to aid in the repair ofcartilage. For example, transforming growth factors beta (TGF-β) havethe capacity to recruit progenitor cells into a chondral defect from thesynovium or elsewhere when loaded in the defect. Hunziker, et al,“Repair of Partial Thickness Defects in Articular Cartilage: CellRecruitment From the Synovial Membrane,” J Bone Joint Surg.,78-A:721-733 (1996). However, the application of growth factors to boneand cartilage implants has not resulted in the expected increase inosteoinductive or chondrogenic activity.

Each of U.S. Pat. Nos. 5,270,300 and 5,041,138, each herein incorporatedby reference, describes a method for treating defects or lesions incartilage that provides a matrix, possibly composed of collagen, withpores large enough to allow cell population and contain growth factors(TGF-β or other factors (such as angiogenesis factors)) appropriate forthe type of tissue desired to be regenerated.

U.S. Patent Publication No. 2003/0044445, herein incorporated byreference, describes an osteogenic composition prepared by a processincluding the steps of subjecting demineralized bone to an extractionmedium to produce an insoluble extraction product and a solubleextraction product, separating the insoluble extraction product and thesoluble extraction product, drying the soluble extraction product toremove all or substantially all of the moisture in the solubleextraction product, and combining the dried soluble extraction productwith demineralized bone particles. This process involves several stepsand is quite laborious. Studies using the process have shown that theformed osteogenic composition does not have appreciably increasedosteoinductive properties when compared to the demineralized boneparticles to which the dried soluble extraction product is added. It wasfurther determined that the demineralized bone from which the extractionproducts are extracted does not exhibit appreciably decreasedosteoinductive properties when compared with its properties prior toextraction. It is thus theorized that the extraction process withdrawsonly a small fraction of available tissue repair factors.

Overall, current bone and cartilage graft formulations have variousdrawbacks. The osteoinductive factors within the matrices can be rapidlydegraded and, thus, factors associated with the matrix are onlyavailable to recruit cells to the site of injury for a short time afterimplantation. Further, when added to a matrix, the active factors oftendo not appreciably increase the osteoinductive activity of the matrixor, at least, do not increase the osteoinductive activity of the matrixas much as is desirable.

Thus, it would be useful to provide increased osteoinductive activityfrom an osteogenic composition in more concentrated form such thatincreased osteoinductive activity can be seen even with little bone voidspace.

BRIEF SUMMARY

Osteoinductive compositions, and implants and methods for theirproduction, are provided. According to certain embodiments, a carrier isexposed to a treatment or condition that increases at least onebiological activity of the carrier. More specifically, osteoinductivefactors are added to the carrier. The biological activities that may beincreased include, but are not limited to, bone forming; bone healing;osteoinductive activity, osteogenic activity, chondrogenic activity,wound healing activity, neurogenic activity, contraction-inducingactivity, mitosisinducing activity, differentiation-inducing activity,chemotactic activity, angiogenic or vasculogenic activity, andexocytosis or endocytosis-inducing activity.

Thus, an osteoinductive composition is provided comprisingosteoinductive factors, such as may be extracted from demineralizedbone, and a carrier. The osteoinductive composition providesconcentrated or enhanced osteoinductive activity. The osteoinductivecomposition is prepared by providing demineralized bone, extractingosteoinductive factors from the demineralized bone, and adding theextracted osteoinductive factors to a carrier. The carrier andosteoinductive factors may form an osteogenic osteoimplant. Theosteoimplant, when implanted in a mammalian body, can induce at thelocus of the implant the full developmental cascade of endochondral boneformation including vascularization, mineralization, and bone marrowdifferentiation. Also, in some embodiments, the osteoinductivecomposition can be used as a delivery device to administer additionalbioactive agents.

The demineralized bone from which the osteoinductive factors areextracted may be provided in any suitable manner. In a onedemineralization procedure, the bone is subjected to an aciddemineralization step followed by a defatting/disinfecting step.

A simple and economically viable method for extracting osteoinductivefactors from bone is provided herein. The method comprises extractingosteoinductive factors such as noncollagenous proteins (includingosteogenic growth factors) from demineralized bone matrix using achaotropic solvent (e.g., 4M guanidine hydrochloride) or a detergent(e.g., 1% sodium dodecylsulfate), removing the chemical used forextraction in an efficient manner that preserves the biological activityof the growth factors, concentrating the biologically active componentsby purifying away nonessential proteins and inhibitors of bonemorphogenetic protein, and combining the protein extracts with abiologically compatible delivery vehicle. Optionally, methods andmaterials for the preservation of activity during storage may beutilized with the present invention. Alternate methods of extractingosteoinductive factors from bone may be used.

Thus, the extracted osteoinductive factors are added to a carrier. Whenthe osteoinductive factors are added to a carrier, the carrier acts as ascaffold and aids in controlling release kinetics. Any suitable shape,size, and porosity of carrier may be used. Suitable carriers includedemineralized bone; surface demineralized bone; mineralized bone;cancellous scaffolds (mineralized or demineralized); particulate,demineralized, guanidine extracted, species-specific (allogenic) bone;specially treated particulate, calcium phosphates, fatty acids, proteinextracted, demineralized, xenogenic bone; collagen; synthetichydroxyapatites; polymers; hydrogels; starches; polyethylene glycol,tricalcium phosphate, sintered hydroxyapatite, settable hydroxyapatite;polylactic acid; tyrosine polycarbonate; calcium sulfate; collagensheets; settable calcium phosphate; settable polymers; polymericcements; settable poly vinyl alcohols; polyurethanes; and otherbiocompatible settable materials. Further, the carrier may comprisecombinations, modifications, or derivatives of these or others.Optionally, xenogenic bone powder carriers also may be treated withproteases such as trypsin. The osteoinductive factors and carrier (ordelivery or support system) together form an osteoimplant useful inclinical applications.

Any suitable method for adding, or dispersing, the osteoinductivefactors to the carrier may be used. Exactly how this occurs caninfluence on the biological activity of the final formulation. Theextracted osteoinductive factors may have been lyophilized, resulting ina powder composition. For obvious reasons, adding a powder to a bonematrix may be challenging. Thus, it may be desirable to process theosteoinductive factors to form a homogenous mixture that may be moreeasily added to a carrier. This can have a significant impact on therelease kinetics of the osteoinductive factors.

Optionally, other additives may be included in the osteoinductivecomposition. For example, radiopaque substances, angiogenesis promotingmaterials, cytokine inhibitors, bioactive agents, othermedically/surgically useful substances, binding agents, or otherosteoinducing agents may be added

The thus formed osteogenic osteoimplant is intended to be applied at abone repair site, or a site for bone augmentation or ectopic boneformation (e.g., lateral spine fusion). Examples include a siteresulting from injury, defect brought about during the course ofsurgery, infection, malignancy or developmental malformation. Theosteoinductive compositions may also be used as drug delivery devices.

This application refers to various patents, patent applications, journalarticles, and other publications, all of which are incorporated hereinby reference. The following documents are incorporated herein byreference: PCT/US04/43999; PCT/US05/003092; US 2003/0143258 A1;PCT/US02/32941; Current Protocols in Molecular Biology, CurrentProtocols in Immunology, Current Protocols in Protein Science, andCurrent Protocols in Cell Biology, John Wiley & Sons, N.Y., edition asof July 2002; Sambrook, Russell, and Sambrook, Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 2001; Rodd 1989 “Chemistry of Carbon Compounds,” vols.1-5 and supps, Elsevier Science Publishers, 1989; “Organic Reactions,”vols 1-40, John Wiley and Sons, New York, N.Y., 1991; March 2001,“Advanced Organic Chemistry,” 5th ed. John Wiley and Sons, New York,N.Y. In the event of a conflict between the specification and any of theincorporated references, the specification shall control. Wherenumerical values herein are expressed as a range, endpoints areincluded.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description is to be regarded as illustrativein nature and not restrictive.

DEFINITIONS

Bioactive Agent or Bioactive Compound is used herein to refer to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. In some embodiments, the bioactive agent is a growthfactor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.A more complete listing of bioactive agents and specific drugs suitablefor use in the present invention may be found in “PharmaceuticalSubstances: Syntheses, Patents, Applications” by Axel Kleemann andJurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals”, Edited by SusanBudavari et al., CRC Press, 1996; and the United StatesPharmacopeia-25/National Formulary-20, published by the United StatesPharmcopeial Convention, Inc., Rockville Md., 2001, each of which isincorporated herein by reference.

Biocompatible, as used herein, is intended to describe materials that,upon administration in vivo, do not induce undesirable long-termeffects.

Bone as used herein refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

Demineralized, as used herein, refers to any material generated byremoving mineral material from tissue, e.g., bone tissue. In certainembodiments, the demineralized compositions described herein includepreparations containing less than 5% calcium and preferably less than 1%calcium by weight. Partially demineralized bone (e.g., preparations withgreater than 5% calcium by weight but containing less than 100% of theoriginal starting amount of calcium) is also considered within the scopeof the invention. In some embodiments, demineralized bone has less than95% of its original mineral content. Demineralized is intended toencompass such expressions as “substantially demineralized,” “partiallydemineralized,” and “fully demineralized.”

Demineralized bone matrix, as used herein, refers to any materialgenerated by removing mineral material from bone tissue. In preferredembodiments, the DBM compositions as used herein include preparationscontaining less than 5% calcium and preferably less than 1% calcium byweight. Partially demineralized bone (e.g., preparations with greaterthan 5% calcium by weight but containing less than 100% of the originalstarting amount of calcium) are also considered within the scope of theinvention.

Osteoconductive is used herein to refer to the ability of anon-osteoinductive substance to serve as a suitable template orsubstance along which bone may grow.

Osteogenic is used herein to refer to the ability of an agent, material,or implant to enhance or accelerate the growth of new bone tissue by oneor more mechanisms such as osteogenesis, osteoconduction, and/orosteoinduction.

Osteoimplant as used herein refers to any bone-derived implant preparedin accordance with the embodiments of this invention and therefore isintended to include expressions such as bone membrane, bone graft, etc.

Osteoinductive, as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Forexample, most osteoinductive materials induce bone formation in athymicrats when assayed according to the method of Edwards et al.,“Osteoinduction of Human Demineralized Bone: Characterization in a RatModel,” Clinical Orthopaedics & Rel. Res., 357:219-228, December 1998,incorporated herein by reference. In other instances, osteoinduction isconsidered to occur through cellular recruitment and induction of therecruited cells to an osteogenic phenotype. Osteoinductivity scorerefers to a score ranging from 0 to 4 as determined according to themethod of Edwards et al. (1998) or an equivalent calibrated test. In themethod of Edwards et al., a score of “0” represents no new boneformation; “1” represents 1%-25% of implant involved in new boneformation; “2” represents 26-50% of implant involved in new boneformation; “3” represents 51%-75% of implant involved in new boneformation; and “4” represents >75% of implant involved in new boneformation. In most instances, the score is assessed 28 days afterimplantation. However, the osteoinductivity score may be obtained atearlier time points such as 7, 14, or 21 days following implantation. Inthese instances it may be desirable to include a normal DBM control suchas DBM powder without a carrier, and if possible, a positive controlsuch as BMP. Occasionally osteoinductivity may also be scored at latertimepoints such as 40, 60, or even 100 days following implantation.Percentage of osteoinductivity refers to an osteoinductivity score at agiven time point expressed as a percentage of activity, of a specifiedreference score.

Proteases, as used herein, are protein-cleaving enzymes that cleavepeptide bonds that link amino acids in protein molecules to generatepeptides and protein fragments. A large collection of proteases andprotease families has been identified. Some exemplary proteases includeserine proteases, aspartyl proteases, acid proteases, alkalineproteases, metalloproteases, carboxypeptidase, aminopeptidase, cysteineprotease, collagenase, etc. An exemplary family of proteases is theproprotein convertase family, which includes furin. Dubois et al.,American Journal of Pathology (2001) 158(l):305316. Members of theproprotein convertase family of proteases are known to proteolyticallyprocess proTGFs and proBMPs to their active mature forms. Dubois et al.,American Journal of Pathology (2001) 158(1):305-316; Cui et al., TheEmbo Journal (1998) 17(16):4735-4743; Cui et al., Genes & Development(2001) 15:2797-2802, each incorporated by reference herein.

Protease inhibitors, as used herein, refers to chemical compoundscapable of inhibiting the enzymatic activity of protein cleaving enzymes(i.e., proteases). The proteases inhibited by these compounds includeserine proteases, acid proteases, metalloproteases (examples of somematrix metalloprotease inhibitors are shown in FIG. 6),carboxypeptidase, aminopeptidase, cysteine protease, etc. The proteaseinhibitor may act specifically to inhibit only a specific protease orclass of proteases, or it may act more generally by inhibiting most ifnot all proteases. Preferred protease inhibitors are protein or peptidebased and are commercially available from chemical companies such asAldrich-Sigma. Protein or peptide-based inhibitors which adhere to theDBM (or calcium phosphate or ceramic carrier) are particularly preferredas they remain associated with the matrix providing a stabilizing effectfor a longer period of time than freely diffusible inhibitors. Examplesof protease inhibitors include aprotinin, 4-(2aminoethyl)benzenesulfonyl fluoride (AEBSF), amastatin-HCl,alphal-antichymotrypsin, antithrombin III, alphal-antitrypsin,4-aminophenylmethane sulfonyl-fluoride (APMSF), arphamenine A,arphamenine B, E-64, bestatin, CA-074, CA-074-Me, calpain inhibitor I,calpain inhibitor II, cathepsin inhibitor, chymostatin,diisopropylfluorophosphate (DFP), dipeptidylpeptidase IV inhibitor,diprotin A, E-64c, E-64d, E-64, ebelactone A, ebelactone B, EGTA,elastatinal, foroxymithine, hirudin, leuhistin, leupeptin,alpha2macroglobulin, phenylmethylsulfonyl fluo4de (PMSF), pepstatin A,phebestin, 1,10phenanthroline, phosphoramidon, chymostatin, benzamidineHCl, antipain, epsilon aminocaproic acid, N-ethylmaleimide, trypsininhibitor, 1-chloro-3-tosylamido-7-amino2-heptanone (TLCK),1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor, andsodium EDTA. Stabilizing agent is any chemical entity that, whenincluded in an inventive composition comprising DBM and/or a growthfactor, enhances the osteoinductivity of the composition as measuredagainst a specified reference sample. In most cases, the referencesample will not contain the stabilizing agent, but in all other respectswill be the same as the composition with stabilizing agent. Thestabilizing agent also generally has little or no osteoinductivity ofits own and works either by increasing the half-life of one or more ofthe active entities within the inventive composition as compared with anotherwise identical composition lacking the stabilizing agent, or byprolonging or delaying the release of an active factor. In certainembodiments, the stabilizing agent may act by providing a barrierbetween proteases and sugar-degrading enzymes thereby protecting theosteoinductive factors found in or on the matrix from degradation and/orrelease. In other embodiments, the stabilizing agent may be a chemicalcompound that inhibits the activity of proteases or sugar-degradingenzymes. In a preferred embodiment, the stabilizing agent retards theaccess of enzymes known to release and solubilize the active factors.Half-life may be determined by immunolgical or enzymatic assay of aspecific factor, either as attached to the matrix or extracted therefrom. Alternatively, measurement of an increase in osteoinductivityhalf-life, or measurement of the enhanced appearance of products of theosteoinductive process (e.g., bone, cartilage or osteogenic cells,products or indicators thereof) is a useful indicator of stabilizingeffects for an enhanced osteoinductive matrix composition. Themeasurement of prolonged or delayed appearance of a strongosteoinductive response will generally be indicative of an increase instability of a factor coupled with a delayed unmasking of the factoractivity.

Superficially demineralized as used herein refers to bone-derivedelements possessing at least about 90 weight percent of their originalinorganic mineral content, the expression “partially demineralized” asused herein refers to bone-derived elements possessing from about 8 toabout 90 weight percent of their original inorganic mineral content andthe expression “fully demineralized” as used herein refers to bonecontaining less than 8% of its original mineral context.

DETAILED DESCRIPTION

I. Introduction

The present invention provides osteoinductive compositions and implantsand methods for their production. According to certain embodiments, acarrier is exposed to a treatment or condition that increases at leastone biological activity of the carrier, as described above. In certainembodiments, the carrier contains peptides or protein fragments thatincrease the osteoinductive or chondrogenic properties of the carrier.Those of ordinary skill will appreciate that a variety of embodiments orversions of the invention are not specifically discussed below but arenonetheless within the scope of the present invention, as defined by theappended claims.

Bone is made up principally of cells, and also of collagen, minerals,and other noncollagenous proteins. Bone matrices can benondemineralized, partially demineralized, demineralized, deorganified,anorganic, or mixtures of these. DBM is comprised principally ofproteins and glycoproteins, collagen being the primary protein componentof DBM. While collagen is relatively stable, normally being degradedonly by the relatively rare collagenase enzymes, various other proteinsand active factors present in DBM are quickly degraded by enzymespresent in the host. These host-derived enzymes include proteases andsugar-degrading enzymes (e.g., endo- and exoglycosidases, glycanases,glycolases, amylase, pectinases, galacatosidases, etc.). Many of theactive growth factors responsible for the osteoinductive activity of DBMexist in cryptic form, in the matrix until activated. Activation caninvolve the change of a pre or pro function of the factor, or release ofthe function from a second factor or entity that binds to the firstgrowth factor. Thus, growth factor proteins in a DBM or added to a DBMmay have a limited osteoinductive effect because they are rapidlyinactivated by the proteolytic environment of the implant site, or evenwithin the DBM itself.

A number of endogenous factors that play important roles in thedevelopment and/or repair of bone and/or cartilage have been identified.BMPs such as BMP-2 and BMP-4 induce differentiation of mesenchymal cellstowards cells of the osteoblastic lineage, thereby increasing the poolof mature cells, and also enhance the functions characteristic ofdifferentiated osteoblasts. Canalis et al., Endocrine Rev.24(2):218-235, 2003. In addition, BMPs induce endochondral ossificationand chondrogenesis. BMPs act by binding to specific receptors, whichresults in phosphorylation of a class of proteins referred to as SMADs.Activated SMADs enter the nucleus, where they regulate transcription ofparticular target genes. BMPs also activate SMAD-independent pathwayssuch as those involving Ras/MAPK signaling. Unlike most BMPs such asBMP-2 and BMP-4, certain BMPs (e.g., BMP-3) act as negative regulators(inhibitors) of osteogenesis. In addition, BMP-1 is distinct bothstructurally and in terms of its mechanism of action from other BMPs,which are members of the TGF-β superfamily. Unlike certain other BMPs(e.g., BMP-2, BMP-4), BMP-1 is not osteoinductive. Instead, BMP-1 is acollagenolytic protein that has also been shown to cleave chordin (anendogenous inhibitor of BMP-2 and BMP-4). Tolloid is a metalloproteasethat is structurally related to BMP-1 and has proteolytic activitytowards chordin. See Canalis, supra, for further details regarding theactivities of BMPs and their roles in osteogenesis and chondrogenesis.

A variety of endogenous inhibitors of BMPs have been discovered inaddition to chordin. These proteins act as BMP antagonists and includepseudoreceptors (e.g., Bambi) that compete with signaling receptors,inhibitory SMADs that block signaling, intracellular binding proteinsthat bind to activating SMADs, factors that induce ubiquitination andproteolysis of activating SMADs, and extracellular proteins that bindBMPs and prevent their binding to signaling receptors. Among theextracellular proteins are noggin, chordin, follistatin, members of theDan/Cerberus family, and twisted gastrulation. These proteins and theirsequences are known and readily available to one of ordinary skill inthe art.

II. Increasing the Biological Activity of a Carrier

Methods for increasing the biologic activity of a bone, cartilage, orother carrier are provided. Osteoinductive osteoimplants are furtherprovided. The osteoinductive osteoimplants comprise carriers andosteoinductive factors, wherein the carrier has increased biologicalactivity relative to a carrier that has not been exposed to a treatmentor condition. The biological activities that may be increased includebut are not limited to osteoinductive activity, osteogenic activity,chondrogenic activity, wound healing activity, neurogenic activity,contraction-inducing activity, mitosis-inducing activity,differentiation-inducing activity, chemotactic activity, angiogenic orvasculogenic activity, and exocytosis or endocytosis-inducing activity.It will be appreciated that bone formation processes frequently includea first stage of cartilage formation that creates the basic shape of thebone, which then becomes mineralized (endochondral bone formation).Thus, in many instances, chondrogenesis may be considered an early stageof osteogenesis, though of course it may also occur in other contexts.

An osteoinductive composition is provided comprising osteoinductivefactors, such as may be extracted from demineralized bone, and acarrier. The osteoinductive composition provides concentrated orenhanced osteoinductive activity. The osteoinductive composition isprepared by providing demineralized bone, extracting osteoinductivefactors from the demineralized bone, and adding the extractedosteoinductive factors to a carrier. The carrier and osteoinductivefactors may form an osteogenic osteoimplant. The osteoimplant, whenimplanted in a mammalian body, can induce at the locus of the implantthe full developmental cascade of endochondral bone formation includingvascularization, mineralization, and bone marrow differentiation. Also,in some embodiments, the osteoinductive composition can be used as adelivery device to administer bioactive agents.

In certain embodiments, the carrier contains peptides or proteinfragments that increase its osteoinductive or chondrogenic properties.The peptides or protein fragments may be exogenously added to thecarrier. Further, other agents may be added to the carrier, e.g., agentsthat improve the osteogenic and/or chondrogenic activity of the matrixby either transcriptional or post-transcriptional regulation of thesynthesis of bone or cartilage enhancing or inhibiting factors by cellswithin the carrier.

III. Provide Demineralized Bone

The demineralized bone from which the osteoinductive factors areextracted may be provided in any suitable manner. The bone useful in theinvention herein is obtained utilizing methods well known in the art,e.g., allogenic donor bone. Bone-derived elements can be readilyobtained from donor bone by various suitable methods, e.g., as describedin U.S. Pat. No. 6,616,698, incorporated herein by reference. The bonemay be cortical, cancellous, or cortico-cancellous of autogenous,allogenic, xenogenic, or transgenic origin.

DBM preparations have been used for many years in orthopedic medicine topromote the formation of bone. For example, DBM has found use in therepair of fractures, in the fusion of vertebrae, in joint replacementsurgery, and in treating bone destruction due to underlying disease suchas rheumatoid arthritis. DBM is thought to promote bone formation invivo by osteoconductive and osteoinductive processes. The osteoinductiveeffect of implanted DBM compositions is thought to result from thepresence of active growth factors present on the isolated collagen-basedmatrix. These factors include members of the TGF-β, IGF, and BMP proteinfamilies. Particular examples of osteoinductive factors include TGF-β,IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenicfactors. Other osteoinductive factors such as osteocalcin andosteopontin are also likely to be present in DBM preparations as well.There are also likely to be other unnamed or undiscovered osteoinductivefactors present in DBM.

In one demineralization procedure, the bone is subjected to an aciddemineralization step followed by a defatting/disinfecting step. Thebone is immersed in acid over time to effect demineralization. Acidsthat can be employed in this step include inorganic acids such ashydrochloric acid and as well as organic acids such as formic acid,acetic acid, peracetic acid, citric acid, propionic acid, etc. The depthof demineralization into the bone surface can be controlled by adjustingthe treatment time, temperature of the demineralizing solution,concentration of the demineralizing solution, and agitation intensityduring treatment.

The demineralized bone is rinsed with sterile water and/or bufferedsolution(s) to remove residual amounts of acid and thereby raise the pH.A suitable defatting/disinfectant solution is an aqueous solution ofethanol, the ethanol being a good solvent for lipids and the water beinga good hydrophilic carrier to enable the solution to penetrate moredeeply into the bone particles. The aqueous ethanol solution alsodisinfects the bone by killing vegetative microorganisms and viruses.Ordinarily, at least about 10 to 40 percent by weight of water (i.e.,about 60 to 90 weight percent of defatting agent such as alcohol) shouldbe present in the defatting disinfecting solution to produce optimallipid removal and disinfection within the shortest period of time. Thepreferred concentration range of the defatting solution is from about 60to about 85 weight percent alcohol and most preferably about 70 weightpercent alcohol.

IV. Extract Osteoinductive Factors from DBM

A simple and economically viable method for extracting osteoinductivefactors from bone is provided herein. The method comprises extractingosteoinductive factors such as noncollagenous proteins (includingosteogenic growth factors) from DBM using a chaotropic solvent or adetergent. The chaotropic solvent may be guanidine hydrochloride of anysuitable concentration, such as 4M. The detergent may be sodiumdodecylsulfate in any suitable concentration, such as 1%. The chemicalused for extraction is removed in an efficient manner that preserves thebiological activity of the growth factors. The biologically activecomponents are concentrated by purifying away nonessential proteins andinhibitors of bone morphogenetic protein, and the protein extracts arethen combined with a biologically compatible delivery vehicle.

Most of the previous work published on the subject uses complicatedextraction schemes that are expensive and difficult to implement in acommercial or industrial application. Using the method described, theprocess is optimized by using less costly chaotropic agents, anddetergents that are easier to remove, than those previously used.Innovative methods to increase the speed of renaturing the extractedproteins are further provided. Typically, dialysis against water is usedto remove the detergent or chaotropic agent. However, by precipitatingthe proteins with ethanol, ammonium sulfate, or polyethylene glycol,dialysis against water is not necessary. Further, ultrafiltration may beused, thereby avoiding dialysis.

Generally, extracted osteoinductive factors have lower specific boneforming activity when compared to the starting material. This may becaused by protein denaturation that results from the extraction. Forexample, when guanidine is used to extract the hydrophobicosteoinductive proteins, the proteins lose their nativethree-dimensional conformation. As a result, unless they regain theirnormal shape upon removal of the guanidine, they no longer are active.The addition of chemical chaperones to the guanidine solution mayprevent this irreversible protein denaturation. Suitable chemicalchaperones include glycerol, trehalose, proline, glycine betaine, anddextrose, along with mixtures of these. These chemical chaperones enablethe osteoinductive proteins to regain their native three-dimensionalconformation when the guanidine is removed. They also prevent proteindenaturation during lyophilization.

Thus, a method for extracting osteoinductive factors from the mineralcomponent of bone is provided to recover growth factor activity that isnormally lost during the demineralization process. It is known that 4 Mguanidine hydrochloride can extract osteoinductive factors from finelypowdered mineralized bone. Additionally, osteoinductive factors can berecovered from the acid that is typically used to demineralized bone,such as 0.6 N HCl. These osteoinductive factors are normally lost duringthe demineralization process and treated as waste.

Growth factors may be extracted from the mineral phase of bone using,for example, the following procedure. As previously described, bone isat least partially demineralized. The bone may comprise powder, fibers,chips, or other. The bone may be demineralized in an acid, for example1M citric acid, 2M citric acid, or 0.6N HCl, at temperatures rangingfrom, for example 1° C. to 28° C. for time period of, for example 10minutes to 96 hours. In one embodiment, the bone is demineralized in anacid at a temperature of 4° C. After demineralization, the acid used fordemineralization contains growth factors and mineral. The acid may bedialyzed against water to cause the mineral phase and the protein growthfactors to co-precipitate. This biphasic (protein and mineral) materialmay then be collected by filtration or centrifutation and combined witha carrier or lyophilized.

In alternative embodiments, the protein and mineral material in the acidmay be separated by dialyzing the acid, also referred to as thedemineralization bath, against a weak acid, for example 0.25M citricacid. In such embodiment, the mineral phase passes through the dialysisbag and the protein phase (collagen fragments, growth factors, etc.) isleft within the bag. The protein phase can then be recovered bydialyzing against water and separating water soluble and water insolubleproteins from one another.

In one embodiment, the method for extracting growth factors comprisesdemineralizing powdered bone with dilute acid within a dialysis bag.Suitable dilute acid includes 0.05 M to 1.0 M HCl and 1M or 2M citricacid. After removing the demineralized bone, the contents of the bag maybe further dialyzed against dilute acid to remove the mineralcomponents. A volatile acid, such as acetic acid, can be used tofacilitate recovery by lyophilization.

Thus, mineralized bone or bone mineral recovered from demineralizationacid may be used as a means of purifying recovered proteins. The proteinphase recovered from the demineralization bath may be solubilized inurea or other form of detergent solution. The bone stimulating growthfactors may then be purified, for example using a hydroxyapatiteaffinity chromatogrphay scheme, described below.

In one embodiment a protein composition substantially free frominorganic components is provided. The protein composition may compriseless than 5% inorganic components by weight. In an alternativeembodiment, a protein composition comprising organic components rangingfrom approximately 6% to approximately 20% by weight is provided. Inanother embodiment, a protein composition comprising organic componentsranging from approximately 21% to approximately 50% may be provided. Inyet a further embodiment, a protein composition comprising organiccomponents ranging from approximately 51% to approximately 90% may beprovided. The protein composition may be recovered from acid used todemineralize bone. The proteinaceous material of the protein compositionmay be purified by chromatography, electrophoresis, or other chemical orphysical means. The protein composition may be combined with anothermaterial such as demineralized bone, hydroxyapatite, tricalciumphosophate,dicalcium phosphate or other. In some embodiments, theprotein composition may exhibit the ability to induce heterotopic boneformation in an athymic animal. In other embodiments the proteincomposition can serve as a source of collagen Type I and otherextracellular matrix proteins that can support tissue repair processessuch as angiogenesis, osteoconduction and wound healing. As the proteinmaterial has desirable handling properties when combined with water orglycerol, the protein can also serve as a carrier for a variety of boneforming matrices including DBM.

In some embodiments, the protein composition may be solubalized in anappropriate medium, such as 6M urea, exposed to hydroxyapatite, TCP,DCP, mineralized bone, surface demineralized bone, or mineral recoveredfrom acid used to demineralize bone. The protein may further bepermitted to adsorb onto the mineral sufaces and be washed with asolution comprising, for example, sodium phosphate ranging fromapproximately 1 mM to 50 mM in concentration. The proteins may then beeluted with a solution comprising, for example, sodium phosophateranging in concentrations from between approximately 100 mM toapproximately 500 mM.

As is described below, growth factors recovered from the mineral phaseof bone may be recombined with an osteoinductive carrier.

Proteases may reduce the activity of the osteoinductive factors indemineralized bone by breaking down those osteoinductive factors. Thisnegative effect may be reduced or eliminated by adding proteaseinhibitors to the HCl solution. Suitable protease inhibitors for use inthe present invention include N-ethyl maleimide, benzamidine HCl,cysteine, or iodoacetic acid. Alternatively, the bone may be heatedbriefly to inactivate the proteases, which are relatively more heatsensitive than the growth factors. A suitable heating regimen is 5minutes at 60° C., or 1 minute at 90° C.

In alternative embodiments, extraction of osteoinductive factors frommineralized or demineralized bone may be done in any suitable manner.Further, during extraction, coprecipitates may be used. Thus, forexample, bone may be treated with a chaotropic solvent such as guanidinehydrochloride. The bone and chaotropic solvent are dialyzed againstwater. As the chaotropic solvent decreases, it is replaced by water.Precipitates are then extracted. Coprecipitates, such as protein,collagen, collagen fragments, albumen, or protein with RGD sequences,may be extracted. The extracted osteoinductive factors andcoprecipitates may then be blended into a homogenous mixture.

Proteins in bone matrix tend to be insoluble and may associate with thebone matrix. Generally, collagens are among the most insolubleosteoinductive factors. Extraction methods may be used to increase thesolubility of the osteoinductive factors to facilitate extraction of theosteoinductive factors. Generally, growth factors are hydrophobic andare not readily soluble. The growth factors may be treated to improvesolubility.

The solubility of the demineralized bone in one or more solvents (e.g.,an aqueous medium) may be changed, e.g., increased, relative, forexample, to the solubility of a standard DBM not exposed to thetreatment. Preferably, the aqueous medium is at physiologicalconditions, e.g., pH, osmotic pressure, salt concentration, etc. arewithin physiologically appropriate ranges. For example, the pH may beapproximately 7.2-8.0, or preferably 7.4-7.6. The osmotic pressure maybe approximately 250-350 mosm/kg, 280-300 mosm/kg, etc. More generally,the pH may be between approximately 3-11, 4-10, 5-9, 6-8.5, etc. Theosmotic pressure may be between 50-500 mosm/kg, 100-350 mosm/kg, etc.The salt concentration may be approximately 100-300 mM NaCl, e.g.,approximately 150 mM NaCl. The aqueous medium may be tissue culturemedium, blood, extracellular fluid, etc., and the physiologicalconditions may be conditions such as are typically found within thesefluids and/or within a body tissue such as muscle. The solubility may beincreased at any temperature, e.g., room temperature, body temperatureof a subject such as a human or animal, etc.

Collagenase treatment of standard human DBM significantly increases itssolubility relative to that of untreated standard human DBM. Thesolubility of the DBM may be increased by exposure to an appropriatetreatment or condition, e.g., collagenase treatment, radiation, heat,etc. The extent to which the solubility is increased may be varied byvarying the nature of the treatment (e.g., the enzyme concentration)and/or the time over which it is applied. A combination of treatmentsmay be used. In certain embodiments of the invention, the solubility ofthe human DBM composition is greater than that of a standard DBMcomposition by between 10% and 4000% percent. For example, thesolubility may be greater by between 10% and 100%, 100% and 500%, 500%and 1000%, 1000% and 2000%, 2000% and 3000%, 3000% and 4000% or anyother range between 10% and 4000%. The solubility may be assessed at anytime following the treatment. For example, the DBM may be placed inaqueous medium for a period of time such as 244-8 hours, 3, 4, 5, 6, or7 days, 10 days, 14 days, etc. The amount of DBM remaining after theperiod of time is quantitated (e.g., dry weight is measured) andcompared with the amount that was present initially. The extent to whichthe amount decreases after a period of time serves as an indicator ofthe extent of solubilization.

Extraction may extract both osteoinductive factors and their inhibitors.If the inhibitors are extracted, it may be desirable in some instancesto separate out the osteoinductive factors. This may be referred to asremoval of the inhibitors or concentration of the osteoinductivefactors.

As a general matter, both the osteoinductive factors and the inhibitorsmay be extracted and both the osteoinductive factors and the inhibitorsmay be used for manufacturing the osteogenic osteoimplant. Alternately,only the osteoinductive factors (and not their inhibitors) are extractedand only the osteoinductive factors are used for manufacturing theosteogenic osteoimplant. Lastly, both the osteoinductive factors and theinhibitors may be extracted and only the osteoinductive factors may beused for manufacturing the osteogenic osteoimplant. The embodiment ofextraction and resultant use of osteoinductive factors with or withoutinhibitors is not a limiting feature of the present invention.

In one embodiment, a simplified extraction process is used that isamenable to batch processing. K. Behnam, E. Brochmann, and S. Murray;Alkali-urea extraction of demineralized bone matrix removes noggin, aninhibitor of bone morphogenetic proteins; Connect Tissue Res. 2004,45(4-5):257-60.

In certain embodiments, the bone is exposed to a treatment or conditionthat generates peptides and protein fragments having osteoinductive orchondrogenic activity. In contrast to various longer proteins, certainpeptides and protein fragments are less susceptible to proteolyticdegradation and more likely to maintain their osteoinductive orchondrogenic properties in the proteolytic environment of the matrix orimplant site. Many osteoinductive and chondrogenic proteins, forexample, growth factors such as BMPs, cell signaling molecules,transcription factors, hormones, etc., have domains that are responsiblefor binding to receptors and/or initiating signal transduction in boneand cartilage growth pathways. These domains are capable of functioningindependently as peptides and protein fragments. In certain embodiments,the osteoinductive or chondrogenic activity of bone and cartilagematrices is increased by cleaving the osteoinductive and chondrogenicfactors present in the matrix to generate active peptides or proteinfragments and/or to generate active peptides or protein fragments thatare less susceptible to degradation than their longer precursors. Theincreased number of factors in the matrix results in increased bone orcartilage formation.

If desired, the osteoinductive factors can be modified in one or moreways, e.g., their protein content can be augmented or modified asdescribed in U.S. Pat. Nos. 4,743,259 and 4,902,296, the contents ofwhich are incorporated by reference herein. As discussed more fullybelow, the osteoinductive factors can be admixed with one or moreoptional substances such as binders, fillers, fibers, meshes, substancesproviding radiopacity, plasticizers, biostatic/biocidal agents, surfaceactive agents, and the like, prior to, during, or after adding to thecarrier.

A number of naturally occurring proteins from bone or recombinantosteoinductive factors have been described in the literature and aresuitable for use in the osteoinductive composition. Recombinantlyproduced osteoinductive factors have been produced by several entities.Creative Biomolecules of Hopkinton, Mass., produces an osteoinductivefactor referred to as Osteogenic Protein 1, or OP1. Genetics Instituteof Cambridge, Mass., produces a series of osteoinductive factorsreferred to as Bone Morphogenetic Proteins 1-13 (i.e., BMP 1-13), someof which are described in U.S. Pat. Nos. 5,106,748 and 5,658,882 and inPCT Publication No. WO 96/39,170, each herein incorporated by reference.Purified osteoinductive factors have been developed by several entities.Collagen Corporation of Palo Alto, Calif., developed a purified proteinmixture that is purported to have osteogenic activity, as described inU.S. Pat. Nos. 4,774,228, 4,774,322, 4,810,691, and 4,843,063, eachherein incorporated by reference. Urist developed a purified proteinmixture which is purported to be osteogenic, as described in U.S. Pat.Nos. 4,455,256, 4,619,989, 4,761,471, 4,789,732, and 4,795,804, eachherein incorporated by reference. International Genetic Engineering,Inc. of Santa Monica, Calif., developed a purified protein mixture thatis purported to be osteogenic, as described in U.S. Pat. No. 4,804,744,herein incorporated by reference.

One osteoinductive factor that may be used in the osteoinductivecomposition is described in detail in U.S. Pat. No. 5,290,763, hereinincorporated by reference. This osteoinductive factor has a highosteogenic activity and degree of purity. The osteoinductive factor ofthe '763 patent exhibits osteoinductive activity at about 3 microgramswhen deposited onto a suitable carrier and implanted subcutaneously intoa rat. In one embodiment, the osteoinductive factor is anosteoinductively active mixture of proteins that exhibit the gelseparation profile shown in FIG. 1 of U.S. Pat. No. 5,563,124, hereinincorporated by reference.

In some embodiments, bone stimulating growth factors, for examplerecovered from the mineral phase of bone, may be purified using aapatite affinity chromatography scheme. Thus, mineralized or surfacedemineralized bone may be used as a chromatography resin. Bone mineralcomprises calcium phosphate sales similar to hydroxyapatite. To usemineralized or surface demineralized bone as a chromatography resin,excess lipid and protein may be removed from the surfaces of the bone.In other embodiments, a similar scheme may be done using demineralizedbone matrix as a resin. In yet further embodiments, recovered inorganicbone mineral (sintered or unsintered) may be used as the chromatographyresin.

In one embodiment, the protocol for such scheme may be as follows.Mineralized bone particles, for example ranging from 100 μm to 5 mm, areprepared. The surface of the mineralized bone particles is cleaned, forexample by soaking or stirring the bone particles in a dilute base suchas 0.1M NaOH for several minutes. Generally, such surface cleaningremoves proteins as well as lipids. In alternative embodiments, surfacecleaning may be performed using supercritical CO₂. Growth factorextracts from the mineral phase may be solubalized in a chaotropicsolvent such as 6M urea. The growth factor solution may then be mixedwith the mineralized bone particles, for example, for several minutes.During such mixing, proteins having an affinity for hydroxyapatite bindto the bone surfaces. The bone-protein complex is then precipitated andthe supernatant removed. The bone-protein complex may be treated toremove weakly bound proteins such as collagen fragments while retainingosteoinductive proteins (the osteoinductive proteins remain bound to thematerial). Such treatment may comprise treating the bone-protein complexwith a 6M urea containing low concentrations of sodium phosphate. Thetreated bone-protein complex may be centrifuged and the supernatantaspirated. In some embodiments, the bone-protein complex may be treatedwith urea containing higher concentrations of sodium phosphate (e.g.,100 mM, 180 mM, or 250 mM) to release bound osteoinductive proteins.Alternatively, the bone-osteoinductive protein complex may belyophilized and formulated with a carrier, for example for orthopedicapplications. Further, the bone protein comples may be used as a growthfactor microcarrier that can be distributed in a DBM macrocarrier.

In yet a further embodiment, an osteoinductive composition with reducedimmunogenicity is provided. The osteoinductive composition comprisesnoncollagenous proteins and mineral recovered acid. The noncollagenousproteins may be extracted from demineralized bone or recovered from acidused to demineralize bone. The mineral recovered acid may be from acidused to demineralize bone. The noncallegnous proteins and mineralrecovered acid may be combined such that the osteoinductive compositionexhibits the ability to induce the formation of heterotopic bone in anormal (euthymic) mouse.

V. Optional Prossing

As mentioned above, in some instances it may be desirable to removeinhibitors or concentrate the osteoinductive factors. This is optionaland may be done by any suitable method. Generally, it may be desirableto remove the inhibitors quickly without denaturing the osteoinductivefactors.

Suitable osteoinductive factors may be obtained by purification ofnaturally occurring proteins from bone or by recombinant DNA techniques.As used herein, the term recombinantly produced osteoinductive factorsrefers to the production of osteoinductive factors using recombinant DNAtechnology. For example, nucleic acids encoding proteins havingosteogenic activity can be identified by producing antibodies that bindto the proteins. The antibodies can be used to isolate, by affinitychromatography, purified populations of a particular osteogenic protein.The amino acid sequence can be identified by sequencing the purifiedprotein. It is possible to synthesize DNA oligonucleotides from theknown amino acid sequence. The oligonucleotides can be used to screeneither a genomic DNA and/or cDNA library made from, for example, bovineDNA, to identify nucleic acids encoding the osteogenic protein. Thecorrect oligonucleotide will hybridize to the appropriate cDNA, therebyidentifying the cDNA encoding the osteogenic protein encoding gene.

In other embodiments, the DBM may include and/or be treated with agentsthat inhibit the activity of one or more activating enzymes, proteases,or glycosidases. Such inhibitory agents are expected to reduce theactivity of specific enzymes (whether derived from the host or from theDBM) that would otherwise interact with osteoinductive agents or otheractive agents in the DBM, thereby reducing osteoinductivity or woundhealing.

The treatment or condition may cleave an inhibitory factor that wouldotherwise inhibit a positively acting agent (by which is meant an agentthat enhances a biological activity of the bone matrix). For example, avariety of proteins or protein fragments are known to inhibit theosteoinductive and/or osteogenic activity of certain bone morphogeneticproteins such as BMP-2. In certain embodiments the inhibitory effect ofa protein or protein fragment is reduced by exposing to a treatment orcondition that causes the cleavage or degradation of the inhibitoryagent. The treatment or condition may block the interaction of theinhibitory agent with its target (e.g., BMP-2) or may inhibit synthesis,secretion, post-translational modification, transport, etc., of theinhibitory agent. For example, the bone may be exposed to antibody to aninhibitory agents or the antibody can be added to the bone.

As will be appreciated by those skilled in the art, factors havingosteoinductive, osteogenic, and/or chondrogenic activity can beinhibited by a variety of mechanisms including proteolytic degradation,binding or sequestration of the factor, etc. A variety of proteins orprotein fragments inhibit the osteoinductive and/or osteogenic activityof certain bone morphogenetic proteins, such as BMPs-2, -4, -5, -6, and-7. Among these inhibitory agents are noggin, chordin, gremlin, Dan,Cerberus, the protein related to Dan and Cerberus (PRDC), caronte,Dante, sclerostin, follistatin, follistatin-related gene (FLRG),ventroptin, and alpha2 HSglycoprotein. Noggin blocks the effect of BMPsin cells of the osteoblastic lineage, and the addition of noggin toosteoblasts in culture blocks BMP-induced synthesis of collagen andnoncollagen proteins, and also inhibits the stimulatory effect of BMPson alkaline phosphatase activity. Chordin acts in a similar fashion.Further details regarding these inhibitory agents are found in Canalis,supra, and references cited therein.

Certain collagen fragments are also believed to inhibit BMPs. Forexample, a potentially inhibitory collagen fragment corresponds to theC-terminal end of procollagen that is released during extracellularmatrix remodeling and collagen assembly. The sequence for a chordin-likecollagen fragment (from Type I collagen) is YVEFQEAGSC VQDGQRYNDKDVWKPEPCRI CVCDTGTVLC DDIICEDVKD CLSPEIPFGECCPICPADLAAAA (SEQ ID NO: 1).

Bone or cartilage inhibitory factors (BCIF) can be inactivated orinhibited by a variety of methods. For example, a specific protease thatcleaves or degrades the BCIF can be used. Another approach is to use anantibody that binds to the BCIF and blocks its interaction with apositively acting factor such as BMP-2 or BMP-4. The antibody mayinhibit post-translational modification, transport, etc., of theinhibitory agent. Antibodies to the inhibitory agents mentioned herein(and others) are known in the art or could be generated using knownmethods without undue experimentation. Either polyclonal or monoclonalantibodies, or antigen-binding fragments thereof, can be used. Otheragents having specific binding ability (e.g., affibodies) could likewisebe used. One of ordinary skill in the art will be able to generateappropriate antibodies, affibodies, etc., based upon the known sequencesof the inhibitory proteins.

VI. Provide a Carrier

Thus, the extracted osteoinductive factors (and possibly inhibitors) maybe added to a carrier. For ease of reference, unless otherwise noted,reference to osteoinductive factors refers to osteoinductive factorswith or without inhibitors. When the osteoinductive factors are added toa carrier, the carrier acts first as a bulking means for applying asmall amount of extracted material. The carrier also may serve as ascaffold, and may aid in controlling release kinetics. Suitable carriersinclude DBM, including surface demineralized bone; mineralized bone;nondemineralized cancellous scaffolds; demineralized cancellousscaffolds; particulate, demineralized, guanidine extracted,species-specific (allogenic) bone; specially treated particulate,protein extracted, demineralized, xenogenic bone; collagen; synthetichydroxyapatites; polymers; hydrogels; starches; polyethylene glycol,tricalcium phosphate, sintered hydroxyapatite, settable hydroxyapatite;polylactic acid; tyrosine polycarbonate; calcium sulfate; collagensheets; settable calcium phosphate; polymeric cements; settable polyvinyl alcohols, polyurethanes; resorbable polymers; polysaccharides andother large polymers; liquid settable polymers; and other biocompatiblesettable materials. Settable materials may be used, and they may set upeither in situ, or prior to implantation. Optionally, xenogenic bonepowder carriers also may be treated with proteases such as trypsin.Preferably, xenogenic carriers are treated with one or more fibrilmodifying agents to increase the intraparticle intrusion volume(porosity) and surface area. Useful agents include solvents such asdichloromethane, trichloroacetic acid, acetonitrile and acids such astrifluoroacetic acid and hydrogen fluoride. The osteoinductive factorsand carrier (or delivery or support system) together form anosteoimplant useful in clinical applications.

Any suitable shape, size, and porosity of carrier may be used. Ratstudies show that the new bone is formed essentially having thedimensions of the device implanted. Generally, particle size influencesthe quantitative response of new bone; particles between 70 μm and 420μm elicit the maximum response. However, other particle sizes may beused. Contamination of the carrier with bone mineral may inhibit boneformation.

In some embodiments, the carrier may be settable and/or injectable. Suchcarrier may be, for example, a polymeric cement, a settable calciumphosphate, a settable poly vinyl alcohol, a polyurethane, or a liquidsettable polymer. Suitable settable calcium phosphates are disclosed inU.S. Pat. Nos. 5,336,264 and 6,953,594, which are hereby incorporated byreference.

A successful carrier for osteoinductive factors performs severalfunctions. It carries osteoinductive factors and allows appropriaterelease kinetics. A successful carrier also appropriately accommodateseach step of the cellular response during bone development, and in somecases protects the osteoinductive factors from nonspecific proteolysis.In addition, selected materials must be biocompatible in vivo andoptionally biodegradable. In some uses, the carrier must act as atemporary scaffold until replaced completely by new bone. Polylacticacid (PLA), polyglycolic acid (PGA), and various combinations havedifferent dissolution rates in vivo. In bone, the dissolution rates canvary according to whether the implant is placed in cortical ortrabecular bone.

The carrier may comprise a shape-retaining solid made of loosely adheredparticulate material, e.g., with collagen. It may also comprise amolded, porous solid, or simply an aggregation of close-packed particlesheld in place by surrounding tissue. Masticated muscle or other tissuemay also be used. Large allogenic bone implants may act as a carrier iftheir marrow cavities are cleaned and packed with particles and theosteoinductive factors.

The osteoimplant resulting from the carrier and the osteoinductivefactors may assume a determined or regular form or configuration such asa sheet, plate, disk, tunnel, cone, or tube, to name but a few.Prefabricated geometry would include, but not be limited to, a crescentapron for single site use, an I-shape to be placed between teeth forintra-bony defects, a rectangular bib for defects involving both thebuccal and lingual alveolar ridges, neutralization plates,reconstructive plates, buttress plates, T-buttress plates, spoon plates,clover leaf plates, condylar plates, compression plates, bridge plates,or wave plates. Partial tubular as well as flat plates can be fabricatedfrom the osteoimplant. Such plates may include such conformations as,e.g., concave contoured, bowl shaped, or defect shaped. The osteoimplantcan be machined or shaped by any suitable mechanical shaping means.Computerized modeling can provide for the intricately-shapedthree-dimensional architecture of an osteoimplant custom-fitted to thebone repair site with great precision.

In one embodiment, the osteoimplant induces endochondral bone formationreliably and reproducibly in a mammalian body. The carrier comprisesparticles of porous materials. The pores must be of a dimension topermit progenitor cell migration into the carrier and subsequentdifferentiation and proliferation. The particle size should be withinthe range of 70 .mu.m-850 μm, preferably 70 μm-420 μm, most preferably150 μm-420 μm. It may be fabricated by close packing particulatematerial into a shape spanning the bone defect, or by otherwisestructuring as desired a material that is biocompatible, and preferablybiodegradable in vivo to serve as a “temporary scaffold” and substratumfor recruitment of migratory progenitor cells, and as a base for theirsubsequent anchoring and proliferation. Useful carrier materials includecollagen; homopolymers or copolymers of glycolic acid, lactic acid, andbutyric acid, including derivatives thereof; and ceramics, such ashydroxyapatite, tricalcium phosphate and other calcium phosphates.Combinations of these carrier materials also may be used.

Mineralized or surface deminralized bone particles may alternatively beused as a carrier. Thus, an osteoinductive composition comprisingmineralized or surface demienralized bone particles and extracts of DBMis provided. The DBM extracts may be adsorbed to the surfaces of themineralized or partially demineralized bone particles. Weakly boundcomponents may be eluted using, for example, low concentrations ofsodium phosphate (for example, 5 mM to 50 mM), thereby concentrating thegrowth factors. In some embodiments, analysis of the proteins bound tothe surfaces of the mineralized or surface demineralized bone particlesindicaes a ratio of Histone H2A to total protein bound elevated by afactor of 2 to 10,000 times over the normal ratio found in extracts ofdemineralized bone matrix or protein recovered from acid used todemineralize bone. In some embodiments, analysis of the proteins boundto the surfaces of the mineralized or surface demineralized boneparticles indicates a ratio of Secreted Phosphoprotein 24 to totalprotein bound elevated by a factor of 2 to 10,000 times over the normalratio found in extracts of demineralized bone matrix or proteinrecovered from acid used to demineralize bone. In some embodiments,analysis of the proteins bound to the surfaces of the mineralized orsurface demineralized bone particles indicates a ratio of BMP-2 to totalprotein bound elevated by a factor of 2 to 10,000 times over the normalratio found in extracts of demineralized bone matrix or proteinrecovered from acid used to demineralize bone. In some embodiments,analysis of the proteins bound to the surfaces of the mineralized orsurface demineralized bone particles indicates a ratio of BMP-4 to totalprotein bound elevated by a factor of 2 to 10,000 times over the normalratio found in extracts of demineralized bone matrix or proteinrecovered from acid used to demineralize bone. In some embodiments,analysis of the proteins bound to the surfaces of the mineralized orsurface demineralized bone particles indicates a ratio of TGF-Beta tototal protein bound elevated by a factor of 2 to 10,000 times over thenormal ratio found in extracts of demineralized bone matrix or proteinrecovered from acid used to demineralize bone.

Use of DBM as Carrier

Any of a variety of DBM preparations may be used as a carrier. DBMprepared by any method may be employed, including particulate orfiber-based preparations, mixtures of fiber and particulatepreparations, fully or partially demineralized preparations, mixtures offully and partially demineralized preparations, and surfacedemineralized preparations. See U.S. Pat. No. 6,326,018, Reddi et al.,Proc. Natl. Acad. Sci. USA (1972) 69:1601-1605; Lewandrowski et al.,Clin. Ortho. Rel. Res., (1995) 317:254-262; Lewandroski et al., J.Biomed. Mater. Res. (1996) 31:365-372; Lewandrowski et al. CalcifiedTiss. Int., (1997) 61:294-297; Lewandrowski et al., I Ortho. Res. (1997)15:748-756, each of which is incorporated herein by reference. Preferreddemineralized bone matrix compositions are described in U.S. Pat. No.5,507,813, herein incorporated by reference. The DBM may be in the formof a section that substantially retains the shape of the original bone(or a portion thereof) from which it was derived. Also useful are DBMpreparations comprising additives or carriers such as polyhydroxycompounds, polysaccharides, glycosaminoglycan proteins, nucleic acids,polymers, polaxomers, resins, clays, calcium salts, and/or derivativesthereof.

The DBM component may be ground or otherwise processed into particles ofan appropriate size before or after demineralization. In certainembodiments, the particle size is greater than 75 microns, morepreferably ranging from about 100 to about 3000 microns, and mostpreferably from about 200 to about 2000 microns. After grinding the DBMcomponent, the mixture may be sieved to select those particles of adesired size. In certain embodiments, the DBM particles may be sievedthough a 50 micron sieve, more preferably a 75 micron sieve, and mostpreferably a 100 micron sieve.

One way to protect small size particles from cellular ingestion and/orprovide a diffusion barrier is to embed them in a monolithicbioabsorbable matrix, and then fragment the particle-containingmonolithic matrix into particle sizes greater than 70 microns,preferably greater than 100 microns, and most preferably greater than150 microns in their smallest dimension. Preferred matrices forembedding small DBM particles include biocompatible polymers and settingcalcium phosphate cements. Generally the particulate DBM/polymer weightratio will range from about 1:5 to about 1:3. In the case of calciumphosphate, the DBM will be present up to 75% by weight. Particulation ofthe monolith can be accomplished by conventional milling or grinding, orthrough the use of cryomilling, or freezing followed by pulverization.In one embodiment, lyophilized DBM is embedded in a resorbable polymer.In a further embodiment, lyophilized DBM is embedded in one of thesetting calcium phosphates known to the art.

Following particulation, the DBM is treated to remove mineral from thebone. While hydrochloric acid is the industry-recognizeddemineralization agent of choice, the literature contains numerousreports of methods for preparing DBM (see, for example, Russell et al.,Orthopaedics 22(5):524-531, May 1999; incorporated herein by reference).Any material that provides a scaffolding containing activeosteoinductive factors is considered DBM. The DBM may be prepared bymethods known in the art or by other methods that can be developed bythose of ordinary skill in the art without undue experimentation. Insome instances, large fragments or even whole bone may be demineralized,and then particulated following demineralization.

In one embodiment, an osteoinductive composition comprising partiallydeminalized bone matrix particles is provided. The partiallydemineralized bone matrix particles may, for example, range in size from500 μm to 4 mm. In one embodiment 10-80% of the mineral of the mineralcontent of the bone is removed. The partially demineralized bone may beheated to temperatures ranging from approximately 40° C. toapproximately 120° C. for period of time ranging from approximately 1minute to approximately 96 hours. Heating may be done with the partiallydemineralized bone in a dry state, in distilled water, in a neutralbuffer solution, or other. The osteoinductive composition may exhibitthe ability to induce the formation of heterotopic bone in a higherorder animal such as a dog, human, or sheep. In some embodiments, theosteoinductive composition may be combined with osteoinductive growthfactors extracted from bone, recovered from acid used to demineralizedbone, or other.

Mixtures of one or more types of demineralized bone-derived elements canbe employed. Moreover, one or more of types of demineralizedbone-derived elements can be employed in combination withnon-demineralized bone-derived elements, i.e., bone-derived elementsthat have not been subjected to a demineralization process. Thus, e.g.,the weight ratio of non-demineralized to demineralized bone elements canbroadly range from about 0:1 to about 1:0. Suitable amounts can bereadily determined by those skilled in the art on a case-by-case basisby routine experimentation.

An osteoinductive demineralized bone matrix scaffold is thus furtherprovided. In one embodiment, the osteoinductive demineralized bonematrix scaffold comprises bone derived components and exhibits, withoutcollagenase pretreatement, the ability to induce specific alkalinephosphotase activity levels higher than those of standard demineralizedbone matrix preparations, for example, 2 to 100,000,000 higher thanstandard dermineralized bone matrix preparations in cultured C2C12cells. In another embodiment, the osteoinductive demineralized bonematrix scaffold comprises osteoinductive growth factors, for exampleextracted from DBM or recovered from acid baths used fordemineralization of the bone matrix, and demineralized bone matrix andexhibits the ability to induce specific alkaline phosphotase activitylevels higher than those of standard demineralized bone matrixpreparations, for example, 2 to 100,000,000 higher than standarddermineralized bone matrix preparations in cultured C2C12 cells.

In addition to the demineralizing step, the bone is optionally subjectedto a configuring step to form an implant. The configuring step can beemployed using conventional equipment known to those skilled in the artto produce a wide variety of geometries, e.g., concave or convexsurfaces, stepped surfaces, cylindrical dowels, wedges, blocks, screws,and the like. A surgically implantable material fabricated fromelongated bone particles that have been demineralized, which may beshaped as a sheet, and processes for fabricating shaped materials fromdemineralized bone particles are disclosed in U.S. Pat. Nos. 5,507,813and 6,436,138, respectively, the contents of which are incorporated byreference herein. Suitable sheets include those sold under the tradename Grafton® DBM Flex, which must be wetted/hydrated prior to use to beuseful for implantation. Such sheets have recently been reported aseffective in seeding human bone marrow stromal cells (BMSCs), which maybe useful in the repair of large bone defects. Kasten et al.,“Comparison of Human Bone Marrow Stromal Cells Seeded onCalcium-Deficient Hydroxyapatite, Betatricalcium Phosphate andDemineralized Bone Matrix,” Biomaterials, 24(15):2593-603, 2003. Alsouseful are demineralized bone and other matrix preparations comprisingadditives or carriers such as binders, fillers, plasticizers, wettingagents, surface active agents, biostatic agents, biocidal agents, andthe like. Some exemplary additives and carriers include polyhydroxycompounds, polysaccharides, glycosaminoglycan proteins, nucleic acids,polymers, polaxomers, resins, clays, calcium salts, and/or derivativesthereof.

The bone used in creating the bone matrix may be obtained from anysource of living or dead tissue. Often, it will be preferred that thesource of bone be matched to the eventual recipient of the inventivecomposition. At a minimum, it is often desirable that the donor andrecipient are of the same species, though even xenogenic sources arepermitted. Thus for use in humans, it is generally preferred to use DBMderived at least in part from human bone. For example, the bone materialmay be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or morehuman bone material. In certain embodiments 100% of the bone material ishuman bone material.

The matrix may be completely insoluble or may be slowly solubilizedafter implantation. Following implantation, preferred matrices resorb ordegrade, remaining substantially intact for at least one to seven days,most preferably for two or four weeks or longer and often longer than 60days. Bioactive agents may be endogenously present in the matrix as inthe case of most demineralized bone, or they may be exogenously added tothe matrix. Matrices may also comprise combinations of endogenous andexogenous bioactive agents.

The matrix may comprise a number of materials in combination, some orall of which may be in the form of fibers and/or particles. The matrixmay comprise calcium phosphates. Driessens et al. “Calcium phosphatebone cements,” Wise, D. L., Ed., Encyclopedic Handbook of Biomaterialsand Bioengineering, Part B, Applications New York: Marcel Decker;Elliott, Structure and Chemistry of the Apatites and Other CalciumPhosphates Elsevier, Amsterdam, 1994, each of which is incorporated byreference. Calcium phosphate matrices include, but are not limited to,dicalcium phosphate dihydrate, monetite, tricalcium phospate,tetracalcium phosphate, hydroxyapatite, nanocrystalline hydroxyapatite,poorly crystalline hydroxyapatite, substituted hydroxyapatite, andcalcium deficient hydroxyapatites.

In one embodiment, an osteoinductive material is provided comprising amineralized particulated material, osteoinductive growth factors, and ademineralized bone matrix scaffold. The mineralized particulatedmaterial may be TCP, hydroxyapatite, mineral recovered from bone,cancellous chips, cortical chips, surface demineralized bone, or othermaterial. The osteoinductive growth factors may be syntheticallyderived, extracted from demineralized bone matrix, recovered fromdemineralization acid bath, or other. The demineralized bone matrixscaffold may be combined with a carrier such as starch or glycerol. Invarious embodiments, the osteoinductive growth factors may besolubilized and combined with the mineralized particulate material, thusallowing adsorption of the growth factors onto the mineral surfaces, aspreviously described. The growth factor/mineral composite may then bedistributed throughout the demineralized bone matrix scaffold. Thegrowth factor/mineral composite thus may comprise a microcarrier and thedemineralized bone matrix scaffold may comprise a macrocarrier. The sizeof the microcarrier mineralized particles may range, for example, from50 nm to 5 mm.

In another embodiment, an osteoinductive composition with reducedimmunogenicity is provided comprising noncollagenous proteins and ademineralized bone matrix scaffold. The noncaollagenous proteins may beextracted, for example, from demineralized bone or recovered from acidused to demineralize bone. The noncollagenous proteins and thedemineralized bone matrix scaffold may be combined such that theosteoinductive composition exhibits the ability to induce the formationof heterotopic bone in a normal (euthymic) mouse.

Treatment of Carrier

In other embodiments, the present invention further provides methods ofincreasing the osteoinductivity of a carrier by exposing the carrier toat least one treatment (e.g., a biological or chemical agent). Inaddition to dispersion of the extracted osteoinductive factors onto thecarrier, the carrier may be exposed to a chemical or condition thatselectively degrades inhibitors of osteogenic activity and/or to achemical or condition that activates osteoinductive factors in thecarrier. Thus, the resulting carrier has an increased osteoinductivity,osteogenic, or chondrogenic activity compared to a similar carrier notexposed to the treatment or condition, because inhibition of anosteoinductive, osteogenic, or chondrogenic factor is blocked. Ingeneral, agents that inhibit or reduce osteoinductive, osteogenic, orchondrogenic activity may be referred to as bone/cartilage inhibitoryfactors (BCIF).

Reduction of Inhibitors

As stated above, the carrier may be treated to reduce inhibitors ofosteoinductive factors. As will be discussed more fully below, theextracted osteoinductive factors are added to the carrier. The additionof the osteoinductive factors and the treatment of the carrier to reduceinhibitors may be performed in combination or sequentially. One or morerounds of treatment may be used, i.e., the treatments may alternate.

Thus, the carrier may be treated to cleave or degrade a specific proteinsuch as an inhibitor of BMP. The carrier is treated such that a specificprotein that is not a major structural component of the carrier isaffected. The specific protein generally makes up less than 1% of thedry weight of the matrix, e.g., less than 0.5%, less than 0.1%, etc. Thespecific protein can be a negatively acting factor, e.g., an inhibitorof a BMP or an inhibitor of a BMP signaling pathway, wherein cleavage ordegradation of the inhibitor allows increased activity of the proteinthat it would otherwise inhibit.

Alternatively or additionally, the carrier may include inhibitory agentspresented in a time-release formulation (e.g., encapsulated in abiodegradable polymer). In the case of activating enzymes, i.e., enzymesthat lead to the release, presentation, or creation of osteoinductivefactors), inhibitory agents that reduce the activity of activatingenzymes preferably lead to increased osteoinductivity over an extendedperiod of time rather than just a burst just after implantation.

Activation

Certain of the osteoinductive or chondrogenic factors found in a bone orcartilage matrix are in cryptic form and must be “activated” or“released” to be osteoinductive. The activation of osteoinductivefactors may involve a conformational change, a post-translationalmodification, protein cleavage, a change in tertiary or quaternarystructure, or release from a binding protein. The osteoinductivefactors, either those extracted and added to the carrier or thosealready present in the carrier, may be in a pre- or pro-form, whichrequires proteolytic cleavage to be active. The osteoinductive factorsalso may be associated with a binding protein or a protein of a bone orcartilage matrix. Proteolysis may also be involved in the activation orinactivation of a binding protein, which could result in activation ofthe osteoinductive peptide or protein fragment. Therefore, alltreatments of a bone or cartilage matrix with any specific ornon-specific condition may affect activation rates of osteoinductivepeptides and protein fragments.

The presence or activation of peptides and/or protein fragments havingosteoinductive or chondrogenic activity may compensate for degradationof osteoinductive or chondrogenic proteins in the carrier, which mayoccur during preparation of the carrier. In certain embodiments it maybe desirable to both inhibit the degradation of osteoinductive orchondrogenic factor and activate or add osteoinductive or chondrogenicfactors such as osteoinductive or chondrogenic peptides or proteinfragments. Variables such as pH and ion concentration may affect proteinfunction and/or folding of the peptide or protein fragment, and mayaffect the activation of osteoinductive or chondrogenic factors. Thesevariables also may affect the release of an osteoinductive factor fromits binding protein. For example, where pH plays a role in theactivation of a factor, the carrier may include a chemical compound suchas a polymer that will break down over time and release an acidby-product; thereby activating the osteoinductive factors within thecarrier. Alternatively, a biodegradable polymer may release ions or aprotease that is able to “activate” the osteoinductive factors of thecarrier.

In certain embodiments, one or more enzymes, such as proteases, lipases,and glycosidases, are added to the carrier to activate theosteoinductive or chondrogenic factors already present (e.g., to convertone or more osteoinductive factors from an inactive to an active form orfrom an active form to a more active form, or from a form that issusceptible to degradation to a form that is less susceptible todegradation, e.g., a form that has a longer half-life).

Many of the growth factors responsible for the osteoinductive orchondrogenic activity of a carrier, such as a bone matrix carrier, existin cryptic form, in the carrier, until activated. Activation can involvethe change of a pre or pro function of a factor, or release of thefunction from a second factor or entity, which binds to the first growthfactor. For example, proteolytic cleavage results in separation of theinactive proprotein (e.g., a proprotein from the TGF superfamily ofproproteins, e.g., TGF-(3) and release of an active, mature peptide). Asproteins of bone and cartilage matrices degrade naturally orartificially, they break down into peptides and protein fragments thatcontain active domains and function as receptor ligands and signaltransducers in bone and cartilage growth signaling pathways. Thesereactions may be promoted to enhance osteoinductive and chondrogenicsignaling in the carrier.

Generate Osteoinductive Peptides and/or Protein Factors in the Carrier

A wide variety of agents, selected from biological agents such asenzymes, chemicals, and conditions, can be used to generateosteoinductive peptides and protein fragments, and these are well knownin the art. The proteases, chemicals, and conditions of the presentinvention can be site specific, amino acid site specific, proteinspecific, semi-site-specific, lipid specific, or sugar specific. Enzymesmay be obtained from endogenous, exogenous, autogenic (autologous),allogenic, or xenogenic sources. They may be purified from naturalsources or produced recombinantly. In many embodiments the enzymes arepurchased from commercial sources (Worthington Biochemical Industries,Sigma, etc.) and either used directly or subsequently purified to befree of contaminants that may negatively affect the activity of thefinal product. Enzymes, peptides, or protein fragments (e.g., generatedby particular proteases) and other treatments may also be purified byconventional methods. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, N.Y.; Ausubel et al. “CurrentProtocols in Molecular Biology”, Greene Publishing Associates, New York,V 1 & 2 1996. Purification can be carried out by a variety ofchromatographic techniques. Size exclusion chromatography is commonlyused. Other methods include ion exchange, hydrophobic interaction, andaffinity chromatography. Peptides or protein fragments may be used inthe bone or cartilage repair matrix as unpurified preparations as longas the peptides or protein fragments maintain their osteoinductive orchondrogenic activity. Alternatively, the enzymes, peptides, or proteinfragments can be synthesized artificially using conventional techniques,produced recombinantly, etc. It may be preferable to use preparationshaving a high degree of purity. For example, an enzyme preparation maycontain at least 90%, at least 95%, at least 98%, at least 99% of theenzyme by weight. The preparation may be essentially free of bacterialcomponents, particularly bacterial components that could causeinflammatory or immunological reactions in a host, such as endotoxin,lipopolysaccharide, etc. Preparations having a purity greater than 99.5%can be used.

One suitable protease is a collagenase. Collagenases and their activityon collagens of various types have been extensively studied. A number ofcollagenase preparations are available from Worthington BiochemicalCorporation, Lakewood, N.J. As described on the company's web site andknown in the art, collagen consists of fibrils composed of laterallyaggregated, polarized tropocollagen molecules (MW 300,000). Eachtropocollagen unit consists of three helically wound polypeptidea-chains around a single axis. The strands have repetitive glycineresidues at every third position and numerous proline and hydroxyprolineresidues, with the particular amino acid sequence being characteristicof the tissue of origin. Tropocollagen units combine uniformly to createan axially repeating periodicity. Cross linkages continue to develop andcollagen becomes progressively more insoluble and resistant to lysis onaging. Gelatin results when soluble tropocollagen is denatured, forexample on mild heating, and the polypeptide chains become randomlydispersed. In this state the strands may readily be cleaved by a widevariety of proteases.

In general, a variety of different collagenases known in the art can beused. Collagenases are classified in section 3.4.24 under theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)enzyme nomenclature recommendations (see, e.g., 3.4.24.3, 3.4.24.7,3,4.24.19). The collagenase can be of eukaryotic (e.g., mammalian) orprokaryotic (bacterial) origin. Bacterial enzymes differ from mammaliancollagenases in that they attack many sites along the helix. Collagenasemay cleave simultaneously across all three chains or attack a singlestrand. Preferably the collagenase cleaves Type I collagen, e.g.,degrades the helical regions in native collagen, preferentially at theY-Gly bond in the sequence Pro-Y-Gly-Pro-, where Y is most frequently aneutral amino acid. This cleavage yields products susceptible to furtherpeptidase digestion. Any protease having one or more of these activitiesassociated with collagenase may be used as a collagenase in accordancewith the present invention.

It will be appreciated that crude collagenase preparations contain notonly several collagenases, but also a sulfhydryl protease, clostripain,a trypsin-like enzyme, and an aminopeptidase. This combination ofcollagenolytic and proteolytic activities is effective at breaking downintercellular matrices, the essential part of tissue disassociation.Crude collagenase is inhibited by metal chelating agents such ascysteine, EDTA, or o-phenanthroline, but not DFP. It is also inhibitedby α2-macroglobulin, a large plasma glycoprotein. Ca²+ is required forenzyme activity. Therefore, it may be desirable to avoid collagenaseinhibiting agents when treating bone matrix with collagenase. Inaddition, although the additional proteases present in some collagenasepreparations may aid in breaking down tissue, they may also causedegradation of desired matrix constituents such as growth factors.Therefore, it may be desirable to use a highly purified collagenase thatcontains minimal secondary proteolytic activities along with highcollagenase activity. For example, a collagenase preparation may containat least 90%, at least 95%, at least 98%, at least 99% collagenase byweight. The preparation may be essentially free of bacterial components,particularly bacterial components that could cause inflammatory orimmunological reactions in a host, such as endotoxin,lipopolysaccharide, etc. Preparations having a purity greater than 99.5%can be used. A suitable preparation is chromatographically purifiedCLSPA collagenase from Worthington Biochemical Corporation. It may bedesirable to include various protease inhibitors that do not inhibitcollagenase but that inhibit various proteases that digest BMP. Forexample, protease inhibitors that are known to protect BMP activity fromdegradation include N-ethyl maleimide, benzamidine hydrochloride,iodoacetic acid, PMSF, AEBSF, E-64. Bestatin may also be used,particularly if the preparation contains aminopeptidase activity. Any ofthese protease inhibitors (or others) can be included in a carrier, suchas a bone matrix composition, or in any composition that is used totreat a carrier.

Another suitable protease bone morphogenetic protein I (BMP-1). BMP-1 isa collagenolytic protein that has also been shown to cleave chordin (aninhibitor of BMP-2 and BMP-4). Thus, BMP-I may be of use to alter thephysical structure of the carrier (e.g., by breaking down collagen)and/or to cleave specific inhibitory protein(s), e.g., chordin ornoggin. Proteins related to any of the proteases described herein, i.e.,proteins or protein fragments having the same cleavage specificity, canalso be used. It will be appreciated that variants having substantialsequence identity to naturally occurring protease can be used. Forexample, variants at least 80% identical over at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100% of the length ofnaturally occurring protease (or any known active fragment thereof thatretains cleavage specificity) when aligned for maximum identity allowinggaps can be used.

Certain preferred proteases include members of the proprotein convertase(PPC) family of proteases, such as furin and related proteases. Membersof this family of cellular enzymes cleave most prohormones andneuropeptide precursors. Numerous other cellular proteins, some viralproteins, and bacterial toxins that are transported by the constitutivesecretory pathway are also targeted for maturation by PCs. Furin andother PC family members share structural similarities that include aheterogeneous ˜10 kDa amino-terminal proregion, a highly conserved ˜55kDa subtilisin-like catalytic domain, and carboxyl-terminal domain thatis heterogeneous in length and sequence. These enzymes becomecatalytically active following proregion cleavage within the appropriatecellular compartment. Furin is the major processing enzyme of thesecretory pathway and is localized in the trans-golgi network, van denOuweland, A. M. W. et al. (1990) Nucl. Acid Res. 18, 664; Steiner, D. F.(1998) Curr. Opin. Chem. Biol. 2, 31-39. Substrates of furin includeblood clotting factors, serum proteins, and growth factor receptors suchas the insulin-like growth factor receptor. Bravo D. A. et al. (1994) J.Biol. Chem. 269, 25830-25873. The minimal cleavage site for furin isArg-X-X-Arg. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg.An additional arginine at the P6 position appears to enhance cleavage.Krysan D. J. et al. (1999) J. Biol. Chem. 274, 2322923234. Furin isinhibited by EGTA, αl-antitrypsin Portland, Jean, F. et al. (1998) Proc.Natl. Acad. Sci. USA 95, 7293-7298, and polyarginine compounds, Cameron,A. et al. (2000) J. Biol. Chem. 275, 36741-36749. Furin has been shownto proteolytically process both proTGF and proBMP proteins, for example,proTGF-βand proBMP-4, respectively, resulting in the release of theactive mature form for each molecule. Dubois et al., American Journal ofPathology (2001) 158(1):305-316; Cui et al., The Embo Journal (1998)17(16):47354743; Cui et al., Genes & Development (2001) 15:2797-2802,each incorporated by reference herein. Furin has also been shown tocleave BMP-2, BMP-6, and BMP-7. For example, furin cleaves between aminoacids 282 and 283 in mature human BMP-2. Newly synthesized human BMP-2contains a signal sequence (amino acids 1-23), a propeptide (amino acids24-282), and an active portion (amino acids 283-396). Furin cleavesmature BMP-2 (amino acids 24-396) between amino acids 282 and 283 torelease the propeptide and the active molecule.

Thus, the carrier, such as a DBM matrix, may be treated with PPCs suchas furin and/or other proteases, which process immature TGF-β and/or BMPsuperfamily propeptides into their active mature forms and/or processactive or inactive TGF-β and/or BMP superfamily polypeptides intosmaller active fragments that are resistant to degradation orinactivation relative to the longer polypeptide, generates a carrierwith increased osteoinductivity compared to a carrier lacking theprotease, resulting in improved bone formation. The higher titers of themature and/or degradation resistant species in these preparationsincrease the osteoinductive capacity of the carrier.

Allogenic cancellous demineralized bone is known not to beosteoinductive. When treated with collagenase enzymes for periodsroutinely used in the art, such as 24 hours, allogenic cancellousdemineralized bone remains nonosteoinductive. Applicants have made thesurprising discovery that allogenic cancellous bone, when treated withcollagenase enzymes for approximately one hour, becomes osteoinductive,approximately as osteoinductive as allogenic cortical demineralizedbone. While not desiring to be bound by any particular scientifictheory, applicants state that it is believed that the disruption of thecollagen matrix makes osteoinductive factors bioavailable. Many types ofcollagenalytic enzymes, such as those set forth herein, would beexpected to render allogenic cancellous demineralized boneosteoinductive when treated as described herein. Other treatments alsoare expected to provide the same result, including the use of salts orionizing or electromagnetic radiation, or various categories of enzymes,so long as the enzymes disrupt the collagen without damaging theosteoinductive factors.

Delivery System

The carrier and the method of adding osteoinductive factors to thecarrier, discussed more fully below, may result in an enhanced deliverysystem. More specifically, the osteoinductive factors may be added to acarrier such that the osteoinductive factors are generally evenlydispersed in three dimensions as opposed to superficially coating acarrier. Dispersion throughout the carrier affects control of releasekinetics of the osteoinductive factors from the carrier. In oneembodiment, a plurality of thin sheets of carrier are provided. Eachsheet of carrier is layered with osteoinductive factors. These sheets ofcarrier layered with osteoinductive factors are stacked.

Thus, optionally, the osteoimplant is formed as a laminate. A laminateosteoimplant may advantageously be shaped in three dimensions, as in theintroduction of a concave surface shape. Further, each layer of thelaminate is continuous, without requiring binding of the joints betweenthe pieces.

Assembling the superimposed layers into a strong unitary structure maybe accomplished by a variety of means/procedures, e.g., application ofknown and conventional biologically compatible adhesives such as thecyanoacrylates; epoxy-based compounds, dental resin sealants, dentalresin cements, glass ionomer cements, polymethyl methacrylate,gelatin-resorcinol-formaldehyde glues, collagen-based glues, inorganicbonding agents such as zinc phosphate, magnesium phosphate or otherphosphate-based cements, zinc carboxylate, etc., and protein-basedbinders such as fibrin glues and mussel-derived adhesive proteins; theuse of mechanical fasteners such as pins, screws, dowels, etc., whichcan be fabricated from natural or synthetic materials and bioabsorbableas well as nonbioabsorbable materials; laser tissue welding; and,ultrasonic bonding. If desired, the layers of the osteogenicosteoimplant can be provided with mechanically interengaging features,e.g., tongue-and-groove, mortise-and-tenon, or dove-tail elements, tofacilitate their assembly into the final product and/or to fix thelayers to each other in a more secure fashion. The optimal method ofassembly would be determined on a case-by-case basis through routineexperimentation. In addition to its carrier and osteoinductive layers,the osteoimplant of this embodiment can optionally possess one or morelayers formed from one or more other materials or substances.

In another embodiment, the carrier may comprise a single thin sheet ofmaterial. The delivery systems thus may comprise a single thin sheet ofmaterial coated with osteoinductive factors. The coated sheet ofmaterial may be rolled or folded over itself such that the growth factorcontent in the interior of the sheet approximates the growth factorlevels at the surfaces.

VII. Dispersion of Osteoinductive Factors onto Carrier

The osteoinductive factors extracted are combined with the appropriatecarrier. Exactly how this occurs can have a significant influence on thebiological activity of the final formulation. The extractedosteoinductive factors may have been lyophilized, resulting in a powdercomposition. In some situations, adding a powder to a bone matrix may bechallenging. Thus, it may be desirable to process the osteoinductivefactors to form a homogenous mixture that may be more easily added to acarrier. This can have a significant impact on the release kinetics ofthe growth factor.

Thus, in a specific example, if the osteoinductive factors arelyophilized and then added to a DBM carrier, the solution is likely tobe inhomogeneous, with most of the osteoinductive factors concentratedon the outside of the DBM carrier. If the osteoinductive factors areadded to a carrier comprising very thin sheets of collagen and thecarrier is then folded in on itself, the distribution of growth factoris more homogenous. The collagen sheets in such an embodiment can bevery thin, on the order of microns.

Any suitable method for adding, or dispersing, the osteoinductivefactors to the carrier may be used. Generally, the procedures used toformulate or disperse osteoinductive factors onto the carrier aresensitive to the physical and chemical state of both the osteoinductivefactors and the carrier. The carriers could potentially be added to theextracts in their denatured state, such as in guanidine, allowing thegrowth factors to be precipitated directly onto the carriers.

In one embodiment, the osteoinductive factors are blended with a bulkingagent to form a homogenous mixture. This mixture is added to thecarrier.

Alternatively, the osteoinductive factors may be blended withcoprecipitates (described more fully above) and this blend may be addedto the carrier. For example, the osteoinductive factor and coprecipitateblend may be added to a carrier such as cancellous chips, syntheticcalcium phosphate, or a liquid settable polymer. Generally, calciumphosphate is a liquid solid slurry while a polymer is a liquid. Thus,the choice of carrier may depend on the desired characteristics of thecomposition. Further, a lubricant, such as water, glycerol, orpolyethylene glycol may be added.

In an alternative embodiment, the extracted osteoinductive factors mayact as their own carrier. In a further embodiment, the aboveosteoimplant can be combined in various ways with other similarosteoimplants or other materials to form an osteoimplant oflaminate-type construction. For example, layers of the osteoimplant ofthe invention herein can be, through chemical or mechanical means,caused to adhere to each other; or, optionally, with other materials,e.g., reinforcing fibers, fabrics, meshes, etc., between some or all ofthe osteoimplant layers. Such laminate materials will differ from knownosteoimplant laminates such as those disclosed in U.S. Pat. No.5,899,939, herein incorporated by reference, in that the final size andarchitecture will be determined by the total amount of starting donormaterial available rather than the specific size or shape of the usabledonor material available.

Formulation

The carrier, the osteoinductive composition, or the osteoimplant may beformulated for a particular use. The formulation may be used to alterthe physical, biological, or chemical properties of the carrier. Aphysician would readily be able to determine the formulation needed fora particular application, taking into account such factors as the typeof injury, the site of injury, the patient's health, and the risk ofinfection. In various embodiments, the osteoinductive composition maycomprise, for example less than approximately 0.5% water, less thanapproximately 1% water, or less than approximately 5% water.

Carriers, osteoinductive compositions, or osteoimplants therefore may beprepared to have selected resorption/loss of osteoinductivity rates, oreven to have different rates in different portions of an implant. Forexample, the formulation process may include the selection of DBMparticles of a particular size or composition, combined with theselection of a particular stabilizing agent or agents, and the amountsof such agents.

In one example, it may be desirable to provide an osteoimplant whoseosteoinductive factors are active in a relatively constant amount over agiven period of time. A DBM carrier comprising factors with longerhalf-lives can be prepared using a less biodegradable polymer or alarger amount (e.g., a thicker coating) of polymeric compound.Alternatively or additionally, the particle size may be important indetermining the half-life of the osteoimplant. In certain embodiments,an inventive formulation may include a mixture of particles, each with adifferent half-life. Such a mixture could provide the steady or possibleunmasking of osteoinductive factors over an extended period of timeranging from days to weeks to months depending on the needs of theinjury. Compositions such as this can be formulated to stimulate bonegrowth in a human patient comparable to the bone growth induced bytreatment with 10 μg of rhBMP on a collagen sponge, and preferablycomparable to 100 μg, and most preferably 1-10 mg rhBMP. When thedegradation of the osteoimplant is of concern, it may be desirable totest the shelf-life of the osteoimplant to determine shelf-life at, forexample, 1, 2, or 3 years. This may be done by storing the osteoimplantat, for example, room temperature or, for accelerated testing, 38degrees Celsius, and periodically checking the inductivity of theosteoimplant. Reference is made to PCT/US05/003092, which is herebyincorporated by reference herein. Implants with enhanced shelf lives mayretain more than about 75% and about 80% of their osteoinductivity afteras long as, or longer than, three years.

Physical properties such as deformability and viscosity of the carriermay also be chosen depending on the particular clinical application. IfDBM is used as a carrier, the particles of the DBM may be mixed withother materials and factors to improve other characteristics of theimplant. For example, the improved DBM material may be mixed with otheragents to improve wound healing. These agents may include drugs,proteins, peptides, polynucleotides, solvents, chemical compounds, andbiological molecules.

Further, the composition may be formulated to be settable and/orinjectable. Thus, for example, the composition may be injectable througha 10-gauge, a 12-gauge, or an 18-gauge needle.

Particles of the carrier may also be formed into various shapes andconfigurations. The particles can be formed into any suitableconfiguration, including rods, strings, sheets, weaves, solids, cones,discs, fibers, and wedges. In certain embodiments, the shape and size ofthe particles in the carrier affect the time course of osteoinductivity.For example, in a cone or wedge shape, the tapered end will result inosteoinductivity shortly after implantation of the osteoimplant, whereasthe thicker end will lead to osteoinductivity later in the healingprocess (hours to days to weeks later). In certain embodiments, theparticle have a length of greater than 2 mm, greater than 1.5 mm,greater than 1 mm, preferably greater than 500 microns, and mostpreferably greater than 200 microns across its widest dimension. Also,larger particle size will have induce bone formation over a longer timecourse than smaller particles. Particles of different characteristics(e.g., composition, size, shape) may be used in the formation of thesedifferent shapes and configurations. For example, in a sheet of DBM alayer of long half-life particles may be alternated between layers ofshorter half-life particles. See U.S. Pat. No. 5,899,939, hereinincorporated by reference, for suitable examples. In a weave, strandscomposed of short half-life particles may be woven together with strandsof longer half-lives.

In one embodiment, fibrous DBM is shaped into a matrix form carrier asdescribed in U.S. Pat. No. 5,507,813, herein incorporated by reference.The shaped DBM is then embedded within a diffusion barrier type matrix,such that a portion of the matrix is left exposed free of the matrixmaterial. Particularly preferred blocking matrices are starch,phosphatidyl choline, tyrosine polycarbonates, tyrosine polyarylates,polylactides, polygalactides, or other resorbable polymers orcopolymers. Devices prepared in this way from these matrices have acombination of immediate and longer lasting osteoinductive propertiesand are particularly useful in promoting bone mass formation in humanposterolateral spine fusion indications.

In another embodiment, carriers having a pre-selected three-dimensionalshape are prepared by repeated application of individual layers of DBM,for example by 3-D printing as described by U.S. Pat. Nos. 5,490,962,5,518,680, and 5,807,437, each incorporated herein by reference.Different layers may comprise individual stabilized DBM preparations, oralternatively may comprise DBM layers treated with stabilizing agentsafter deposition of multiple layers.

In the process of preparing the osteoimplant, the materials may beproduced entirely aseptically or be sterilized to eliminate anyinfectious agents such as HIV, hepatitis B, or hepatitis C. Thesterilization may be accomplished using antibiotics, irradiation,chemical sterilization (e.g., ethylene oxide), or thermal sterilization.Other methods known in the art of preparing DBM such as defatting,sonication, and lyophilization may also be used in preparing a DBMcarrier. Since the biological activity of demineralized bone is known tobe detrimentally affected by most terminal sterilization processes, caremust be taken when sterilizing the inventive compositions.

VIII. Optional Additives

Optionally, other additives may be included in the osteoinductive bonematrix. It will be appreciated that the amount of additive used willvary depending upon the type of additive, the specific activity of theparticular additive preparation employed, and the intended use of thecomposition. The desired amount is readily determinable by the user. Anyof a variety of medically and/or surgically useful optional substancescan be incorporated in, or associated with, the osteoinductive factorseither before, during, or after preparation of the osteogeniccomposition.

In certain embodiments, the additive is adsorbed to or otherwiseassociated with the osteoimplant. The additive may be associated withthe osteoimplant through specific or non-specific interactions, orcovalent or noncovalent interactions. Examples of specific interactionsinclude those between a ligand and a receptor, an epitope and anantibody, etc. Examples of nonspecific interactions include hydrophobicinteractions, electrostatic interactions, magnetic interactions, dipoleinteractions, van der Waals interactions, hydrogen bonding, etc. Incertain embodiments, the additive is attached to the osteoimplant, forexample, to the carrier, using a linker so that the additive is free toassociate with its receptor or site of action in vivo. In otherembodiments the additive is either covalently or non-covalently attachedto the carrier. In certain embodiments, the additive may be attached toa chemical compound such as a peptide that is recognized by the carrier.In another embodiment, the additive is attached to an antibody, orfragment thereof, that recognizes an epitope found within the carrier.In certain embodiments at least additives are attached to theosteoimplant. In other embodiments at least three additives are attachedto the osteoimplant. An additive may be provided within the osteoimplantin a sustained release format. For example, the additive may beencapsulated within biodegradable nanospheres, microspheres, etc.

It will be understood by those skilled in the art that the lists ofoptional substances herewith included are not intended to be exhaustiveand that other materials may be admixed with bone-derived elementswithin the practice of the present invention.

Radiopaque Substances

Radiopaque substances may be added to impart radiopacity to thecomposition. Examples of substances imparting radiopacity include forexample, fully mineralized bone particles, Barium and Iodine containingcompounds or compositions, e.g., Barium Sulfate and Barium Sulfate forSuspension, lopanoic Acid, and the like. When employed, substancesimparting radiopacity will typically represent from about 1 to about 25weight percent of the bone particle containing composition, calculatedprior to forming the shaped material.

Angiogenesis Promoting Materials

Development of a vasculature around the implant site may also beimportant to forming new bone and/or cartilage tissues. Angiogenesis maybe an important contributing factor for the replacement of new bone andcartilage tissues. In certain embodiments, angiogenesis is promoted sothat blood vessels are formed at the site to allow efficient transportof oxygen and other nutrients and growth factors to the developing boneor cartilage tissue. Thus, angiogenesis promoting factors may beincluded in the osteoimplant to increase angiogenesis in that region.For example, class 3 semaphorins, e.g., SEMA3, controls vascularmorphogenesis by inhibiting integrin function in the vascular system,Serini et al., Nature, (July 2003) 424:391-397, incorporated herein byreference, and may be included in the osteoimplant.

Bioactive Agents

The osteoconductive composition may provide a system for deliveringbioactive agents, such as osteoinductive factors, to a host animal.Thus, the osteoimplant enables an improved healing response to theimplant without the need to administer separately the bioactive agent. Aproblem with the introduction of the bioactive agent at the site is thatit is often diluted and redistributed during the healing process by thecirculatory systems (e.g., blood, lymph) of the recipient beforecomplete healing has occurred. A solution to this problem ofredistribution is to affix the bioactive components to the osteoimplant.Some preferred bioactive agents that can be delivered using a DBMcomposition include agents that promote the natural healing process,i.e., resorption, vascularization, angiogenesis, new growth, etc. In oneembodiment, the osteoimplant is provided in which DBM, together with astabilizing agent, is used to deliver the biologically active agent. Itis expected that the stabilizing agent will protect the biologicallyactive agent from degradation, and therefore will extend its active lifeafter delivery into the recipient animal. In certain embodiments, thebioactive agent is an osteoinductive agent, and in certain embodiments,the DBM may be used to deliver more than one bioactive agent, preferablymore than two, and more preferably sometimes more than three bioactiveagents. The bioactive agent may be associated with the DBM. For example,the bioactive agent may be associated with the DBM through electrostaticinteractions, hydrogen bonding, pi stacking, hydrophobic interactions,van der Waals interactions, etc. In certain embodiments, the bioactiveagent is attached to the DBM through specific interactions such as thosebetween a receptor and its ligand or between an antibody and itsantigen. In other embodiments, the bioactive agent is attached to theDBM through non-specific interactions (e.g., hydrophobic interactions).

Medically/surgically useful substances include physiologically orpharmacologically active substances that act locally or systemically inthe host. Generally, these substances may include bioactive substanceswhich can be readily incorporated into the osteoimplant and include,e.g., demineralized bone powder as described in U.S. Pat. No. 5,073,373,the contents of which are incorporated herein by reference; collagen,insoluble collagen derivatives, etc., and soluble solids and/or liquidsdissolved therein; antiviricides, particularly those effective againstHIV and hepatitis; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymycin B,tetracyclines, biomycin, chloromycetin, and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc.;biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids;peptides; vitamins; inorganic elements; co-factors for proteinsynthesis; hormones; endocrine tissue or tissue fragments; synthesizers;enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases,etc.; polymer cell scaffolds with parenchymal cells; angiogenic agentsand polymeric carriers containing such agents; collagen lattices;antigenic agents; cytoskeletal agents; cartilage fragments; living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells; naturalextracts; genetically engineered living cells or otherwise modifiedliving cells; expanded or cultured cells; DNA delivered by plasmid,viral vectors or other means; tissue transplants; demineralized bonepowder; autogenous tissues such as blood, serum, soft tissue, bonemarrow, etc.; bioadhesives; bone morphogenic proteins (BMPs);osteoinductive factor (IFO); fibronectin (FN); endothelial cell growthfactor (ECGF); vascular endothelial growth factor (VEGF); cementumattachment extracts (CAE); ketanserin; human growth hormone (HGH);animal growth hormones; epidermal growth factor (EGF); interleukins,e.g., interleukin-1 (IL-1), interleukin-2 (IL-2); human alpha thrombin;transforming growth factor (TGF-beta); insulin-like growth factors(IGF-1, IGF-2); platelet derived growth factors (PDGF); fibroblastgrowth factors (FGF, BFGF, etc.); periodontal ligament chemotacticfactor (PDLGF); enamel matrix proteins; growth and differentiationfactors (GDF); hedgehog family of proteins; protein receptor molecules;small peptides derived from growth factors above; bone promoters;cytokines; somatotropin; bone digesters; antitumor agents; cellularattractants and attachment agents; immuno-suppressants; permeationenhancers, e.g., fatty acid esters such as laureate, myristate andstearate monoesters of polyethylene glycol, enamine derivatives,alpha-keto aldehydes, etc.; and nucleic acids. The amounts of suchoptionally added substances can vary widely with optimum levels beingreadily determined in a specific case by routine experimentation.

In certain embodiments, the agent to be delivered is adsorbed to orotherwise associated with the osteoimplant. The agent may be associatedwith the osteoimplant through specific or non-specific interactions; orcovalent or non-covalent interactions. Examples of specific interactionsinclude those between a ligand and a receptor, a epitope and anantibody, etc. Examples of non-specific interactions include hydrophobicinteractions, electrostatic interactions, magnetic interactions, dipoleinteractions, van der Waals interactions, hydrogen bonding, etc. Incertain embodiments, the agent is attached to the osteoimplant using alinker so that the agent is free to associate with its receptor or siteof action in vivo. In certain embodiments, the agent to be delivered maybe attached to a chemical compound such as a peptide that is recognizedby the matrix of the DBM composition. In another embodiment, the agentto be delivered is attached to an antibody, or fragment thereof, thatrecognizes an epitope found within the matrix of the DBM composition. Ina further embodiment, the agent is a BMP, TGF-β, IGF, parathyroidhormone (PTH), growth factors, or angiogenic factors. In certainembodiments at least two bioactive agents are attached to the DBMcomposition. In other embodiments at least three bioactive agents areattached to the DBM composition.

Osteoinducing Agents

Other osteoinducing agents besides the extracted osteoinductive factorsmay be added to the carrier. These agents may be added in an activatedor non-activated form. These agents may be added at anytime during thepreparation of the inventive material. For example, in the case of a DBMcarrier, the osteoinducing agent may be added after the demineralizationstep and prior to the addition of the stabilizing agents so that theadded osteoinducing agent is protected from exogenous degrading enzymesonce implanted. In some embodiments the DBM is lyophilized in a solutioncontaining the osteoinducing agent. In certain other embodiments, theosteoinducing agents are adhered onto the hydrated demineralized bonematrix and are not freely soluble. In other instances, the osteoinducingagent is added after addition of a stabilizing agent so that theosteoinducing agent is available immediately upon implantation of theDBM.

Osteoinducing agents include any agent that leads to or enhances theformation of bone. The osteoinducing agent may do this in any manner,for example, the agent may lead to the recruitment of cells responsiblefor bone formation, the agent may lead to the secretion of matrix whichmay subsequently undergo mineralization, the agent may lead to thedecreased resorption of bone, etc. Suitable osteoinducing agents includebone morphogenic proteins (BMPs), transforming growth factor (TGF-0),insulin-like growth factor (IGF-1), parathyroid hormone (PTH), andangiogenic factors such as VEGF. In one embodiment, the inducing agentis genetically engineered to comprise an amino acid sequence whichpromotes the binding of the inducing agent to the DBM or the carrier.Sebald et al., PCT/EPOO/00637, incorporated herein by reference,describe the production of exemplary engineered growth factors suitablefor use with DBM.

VIII. Treatment of Compositions

In the process of preparing improved inventive bone and cartilage matrixmaterials, the materials may be produced entirely aseptically or besterilized to eliminate any infectious agents such as HIV, hepatitis B,or hepatitis C. The sterilization may be accomplished using antibiotics,irradiation, chemical sterilization (e.g., ethylene oxide), or thermalsterilization. Other methods known in the art of preparing bone andcartilage matrices, such as defatting, sonication, and lyophilizationmay also be used in preparing the carrier. Since the biological activityof various materials including demineralized bone is known to bedetrimentally affected by most terminal sterilization processes, caremust be taken when sterilizing the inventive compositions. In someembodiments, the osteoimplants described herein will be preparedaseptically or sterilized.

IX. Example Compositions

In one embodiment, the osteoimplant comprises a combination of DBM andosteoinductive factors from bone that has 2 to 150 fold greater activitythan DBM that has not been supplemented, as measured by the ability ofthe composition to induce heterotopic bone formation in a rat or mouse.Various ratios of DBM to osteoinductive factors may be used, rangingfrom 1 gram DBM:1 ug osteoinductive factors to 1 gram DBM:100 mgosteoinductive factors. The osteoimplant may comprise osteoinductivegrowth factors extracted from DBM or recovered from acid baths used fordemineralization of bone matrix and demineralized bone matrix and mayexhibit the ability to induce specific alkaline phosphatase activityhigher than those of standard demineralized bone matrix preparations,for example, 2 to 100,000,000 higher than standard dermineralized bonematrix preparations in cultured C2C12.

In another embodiment, the osteoimplant comprises a combination of DBM,osteoinductive factors, and a carrier in various ratios. Any suitablecarrier may be used, including polyethylene glycol, lecithin, starch,collagen, hydroxyapatite, or hyalurounic acid. Suitable ratios include 1gram DBM:10 ug to 100 mg osteoinductive factors:100 mg to 10 gramscarrier.

In a further embodiment, the osteoimplant comprises a combination ofnondemineralized bone with osteoinductive factors from bone that has theability to induce posterolateral spine fusion in higher animals, such ashumans and dogs, without the addition of recombinant growth factors oriliac crest autograft. Various ratios of bone to osteoinductive factorsranging from 1 gram bone:1 ug osteoinductive factors to 1 gram bone:100mg osteoinductive factors can be used.

In yet a further embodiment, the osteoimplant comprises a combination ofmineralized bone, osteoinductive factors, and a carrier in variousratios. Any suitable carrier may be used, as set forth above. Suitableratios include 1 gram DBM:10 ug to 100 mg osteoinductive factors:100 mgto 10 grams carrier, with the ability to induce posterolateral spinefusion in humans or dogs without the addition of recombinant growthfactors or iliac crest autograft.

As previously described, in another embodiment, an osteoinductivematerial comprising a mineralized particulated material, osteoinductivegrowth factors, and a demineralized bone matrix scaffold is provided.

In a further previously described embodiment, an osteoinductivecomposition comprising noncollagenous proteins extracted fromdemineralized bone or recovered from acid used to demineralize bone andmineral recovered acid used to demineralize bone. Alternatively, theosteoinductive composition may comprise noncollagenous proteinsextracted from demineralized bone or recovered from acid used todemineralize bone and a demineralized bone matrix scaffold.

In yet a further previously described embodiment, an osteoinductivecomposition comprising mineralized or surface demineralized boneparticles and extracts of DBM may be provided.

X. Assessment of Osteogenic Activity

Induction of bone formation can be determined by a histologicalevaluation showing the de novo formation of bone with accompanyingosteoblasts, osteoclasts, and osteoid matrix. For example,osteoinductive activity of an osteoinductive factor can be demonstratedby a test using a substrate onto which material to be tested isdeposited. The substrate with deposited material is implantedsubcutaneously in a test animal. The implant is subsequently removed andexamined microscopically for the presence of bone formation includingthe presence of osteoblasts, osteoclasts, and osteoid matrix. A suitableprocedure for assessing osteoinductive activity is illustrated inExample 5 of U.S. Pat. No. 5,290,763, herein incorporated by reference.Although there is no generally accepted scale of evaluating the degreeof osteogenic activity, certain factors are widely recognized asindicating bone formation. Such factors are referenced in the scale of0-8 which is provided in Table 3 of example 1 of U.S. Pat. No.5,563,124, herein incorporated by reference. The 0-4 portion of thisscale corresponds to the scoring system described in U.S. Pat. No.5,290,763, which is limited to scores of 0-4. The remaining portion ofthe scale, scores 5-8, references additional levels of maturation ofbone formation. The expanded scale also includes consideration ofresorption of collagen, a factor which is not described in the '763patent.

In studies, a typical amount of DBM for implantation is 20 mg in a mouseand 40 mg in a rat. Significant increases in the growth factor dose, forexample, 150× dose (or 150 times the growth factor found in normal DBM),lead to significantly more and potentially faster bone growth withlarger volume bone growth, more dense bone growth, larger nodules ofbone growth, higher x-ray density, and, generally, a higherosteoinductive score. Associated with this increase in osteoinductivitycan be a cortical shell surrounding the nodule and some level ofvascularization in the nodule. However, the ability to quantitativelymeasure is generally limited by the method used, and generally measuredincreases in osteoinductive activity are not linear with the increase indosage. Thus, if 20 mg of DBM gives an osteoinductive activity of 1, 100times the growth factor dose (2000 mg of DBM growth factors) does notgive an osteoinductive activity of 100. Instead, it may result in anosteoinductive activity of about 20. A limitation of measurement usingosteoinductive scores is that, in some situations, the system's abilityto respond may be saturated. Thus, for example, if the score ranges onlyfrom 1 to 4, two samples may have the same score (4) but may not, infact, be comparable. This is particularly the case when the boneresulting from one method or implant is qualitatively better than thebone resulting from another method or implant. That is, both methods orimplants may result in an osteoinductive score of 4 but one may resultin qualitatively better bone than the other. Thus, in some situations itmay be desirable to test speed of growth, density, presence of corticalbone, shelling, and/or other factors showing an increase over normaldemineralized bone matrix. Further, in addition to, or in lieu of,testing at 28 days, it may be desirable to test inductivity at 21 daysGenerally, inductivity may be measured histomorphometrically by methodsknown in art.

Further, delivering 100 times the growth factor dose may be challenging.In filling a bone defect, only as much filler may be used as there isbone void space.

XI. Uses

Therapeutic Uses

The osteogenic osteoimplant is intended to be applied at a bone repairsite, for example, a site resulting from injury, defect brought aboutduring the course of surgery, infection, malignancy, or developmentalmalformation. The osteoimplant can be utilized in a wide variety oforthopedic, periodontal, neurosurgical, and oral and maxillofacialsurgical procedures.

At the time just prior to when the osteoimplant of the invention is tobe placed in a defect site, optional materials, e.g., autograft bonemarrow aspirate, autograft bone, preparations of selected autograftcells, autograft cells containing genes encoding bone promoting action,etc., can be combined with the osteoimplant. The osteoimplant can beimplanted at the bone repair site, if desired, using any suitableaffixation means, e.g., sutures, staples, bioadhesives, screws, pins,rivets, other fasteners and the like or it may be retained in place bythe closing of the soft tissues around it.

The osteoinductive compositions may also be used as drug deliverydevices. In certain embodiments, association with the osteoinductivecompositions increases the half-life of the relevant biologically activeagent(s). Particularly preferred inventive drug delivery devices areused to deliver osteoinductive growth factors. Other preferred agents tobe delivered include factors or agents that promote wound healing.However, the osteoinductive compositions may alternatively oradditionally be used to deliver other pharmaceutical agents includingantibiotics, anti-neoplastic agents, growth factors, hematopoieticfactors, nutrients, an other bioactive agents described above. Theamount of the bioactive agent included with the DBM composition can varywidely and will depend on such factors as the agent being delivered, thesite of administration, and the patient's physiological condition. Theoptimum levels is determined in a specific case based upon the intendeduse of the implant.

Non-Therapeutic Uses

In addition to therapeutic uses involving implantation into a subject,the osteoinductive compositions have a number of other uses. Forexample, they can be used to generate cell lines, tissues, or organshaving osteogenic or chondrogenic properties. In particular, cells canbe removed from a donor and cultured in the presence of anosteoinductive composition. The invention includes such cells as well astissues and organs derived therefrom. The cells, tissues, or organs maybe implanted into the original donor after a period of culture in vitroor may be implanted into a different subject.

XII. Conclusion

In certain embodiments, the osteoinductive compositions and associatedosteoimplants produce bone or cartilage in an animal model and/or inhuman patients with similar timing and at a level at least 10%, 20%,35%, 50%, 100%, 200%, 300%, or 400% or greater osteogenic,osteoinductive or chondrogenic activity than a corollary carrier thathas not been exposed to a treatment or condition as described herein. Ofcourse, one skilled in the art will appreciate that these values mayvary slightly depending on the type of test used to measure theosteoinductivity or osteogenic or chondrogenic activity described above.The test results may fall within the range of 10% to 35%, 35% to 50%,50% to 100%, 100% to 200%, and 200% to 400%. In certain embodiments,when an osteoimplant is implanted into a bone defect site, theosteoimplant has an osteoinductivity score of at least 1, 2, 3, or 4 inan animal model and/or in humans.

Although the invention has been described with reference to preferredembodiments, persons skilled in the art will recognize that changes maybe made in form and detail without departing from the spirit and scopeof the invention.

1. An osteoinductive composition comprising: a carrier; osteoinductivefactors, the osteoinductive factors being supported by the carrier;wherein the osteoinductive composition exhibits increased osteoinductiveactivity when compared to a carrier not supporting osteoinductivefactors.
 2. The composition of claim 1, wherein the osteoinductivefactors are extracted from demineralized bone.
 3. The composition ofclaim 1, wherein the osteinductive factors are recovered from acid usedto demineralize bone.
 4. The composition of claim 1, wherein theosteoinductive factors comprise noncollagenous proteins.
 5. Thecomposition of claim 1, further comprising a coprecipitate, wherein thecoprecipitate and the osteoinductive factors form a homogenous mixturesupported by the carrier.
 6. The composition of claim 5, wherein thecoprecipitate comprises collagen.
 7. The composition of claim 5, whereinthe coprecipitate comprises hydroxyapatite.
 8. The composition of claim5, wherein the coprecipitate comprises tricalcium phosphate.
 9. Thecomposition of claim 5, wherein the coprecipitate comprises mineralcomponents of bone recovered during demineralization.
 10. Thecomposition of claim 5, wherein the coprecipitate comprises mineralizedfragments of cortical or cancellous bone.
 11. The composition of claim5, wherein the coprecipitate comprises partially demineralized bone. 12.The composition of claim 5, wherein the coprecipitate is selected fromthe group consisting of a protein, a collagenous fragment, albumen,starch, carbodydrate, proteoglycans, heparin sulfate, concanavalin,calmodulin, polyethylene glycol, hyaluronic acid, proteoglycans,hydrophobic resins and a protein with RGD sequences.
 13. The compositionof claim 1, further comprising a bioactive agent, wherein theosteoinductive carrier acts as a delivery device to administer thebioactive agent.
 14. The composition of claim 1, and further comprisinga bioactive agent.
 15. The composition of claim 14, wherein thebioactive agent is selected from the group consisting of osteogenic orchondrogenic proteins or peptides, anti-AIDS substances, anti-cancersubstances, antibiotics, immunosuppressants, anti-viral substances,enzyme inhibitors, hormones, neurotoxins, opioids, hypnotics,anti-histamines, lubricants, tranquilizers, anti-convulsants, musclerelaxants and anti-Parkinson substances, anti-spasmodics and musclecontractants including channel blockers, miotics and anti-cholinergics,anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds,modulators of cell-extracellular matrix interactions including cellgrowth inhibitors and antiadhesion molecules, vasodilating agents,inhibitors of DNA, RNA or protein synthesis, anti-hypertensives,analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatoryagents, anti-angiogenic factors, angiogenic factors, anti-secretoryfactors, anticoagulants and/or antithrombotic agents, local anesthetics,ophthalmics, prostaglandins, anti-depressants, anti-psychoticsubstances, anti-emetics, and imaging agents.
 16. The composition ofclaim 1, wherein the carrier is demineralized bone.
 17. The compositionof claim 1, wherein the carrier is surface demineralized bone.
 18. Thecomposition of claim 1, wherein the carrier is mineralized bone.
 19. Thecomposition of claim 1, wherein the carrier is collagen.
 20. Thecomposition of claim 1, wherein the carrier is mineral recovered frombone.
 21. The composition of claim 1, wherein the carrier ispolyethylene glycol.
 22. The composition of claim 1, wherein the carrieris selected from the group consisting of cancellous scaffolds(mineralized or demineralized); particulate, demineralized, guanidineextracted, species-specific (allogenic) bone; specially treatedparticulate, protein extracted, demineralized, xenogenic bone; synthetichydroxyapatites; polymers; hydrogels; starches; tricalcium phosphate,sintered hydroxyapatite, settable hydroxyapatite; polylactic acid;tyrosine polycarbonate; calcium sulfate; collagen sheets; settablecalcium phosphate; polymeric cements; settable poly vinyl alcohols; andpolyurethanes.
 23. The composition of claim 1, wherein the compositionis settable.
 24. The composition of claim 1, wherein the composition isinjectable.
 25. The composition of claim 1, wherein the carrier andosteoinductive factors comprise a delivery system.
 26. The compositionof claim 25, wherein the carrier comprises a plurality of sheets, eachsheet being coated with osteoinductive factors, wherein the sheets arestacked, thereby forming a laminate.
 27. The composition of claim 26,further comprising an adhesive layer between each sheet.
 28. Thecomposition of claim 25, wherein the carrier comprises a sheet, thesheet being coated with osteoinductive factors, wherein the sheet isrolled or folded such that a growth factor content in an interior of thesheet approximates a growth factor content at a surface of the sheet.29. The composition of claim 1, wherein the osteoinductive factorscomprise an homogenous mixture.
 30. The composition of claim 29, whereinthe homogenous mixture further comprises a bulking agent.
 31. Theosteoinductive composition of claim 1, wherein the carrier isdemineralized bone matrix and the osteoinductive factors areosteoinductive growth factors extracted from DBM or recovered from acidbaths used for demineralization of bone matrix, and wherein the growthfactors and the demineralized bone matrix are combined such that theosteoinductive composition exhibits an ability to induce specificalkaline phosphatase activity levels 2 to 100,000,000 times higher thanstandard demineralized bone matrix preparations in cultured C2C12 cells32. The osteoinductive composition of claim 1, wherein the carriercomprises mineralized or surface demineralized bone particles andwherein the osteoinductive factors comprise extracts of DBM, wherein theextracts of DBM are adsorbed to surfaces of the mineralized or partiallydemineralized bone particles and wherein weakly bound extracts areeluted.
 33. An osteoinductive material comprising: a mineralizedparticulated material; osteoinductive growth factors; and ademineralized bone matrix scaffold; wherein the osteoinductive growthfactors are solubilized and combined with the mineralized particledmaterial to form a growth factor/mineral composite and wherein thegrowth factor/mineral composite is distributed in the demineralized bonematrix scaffold.
 34. The osteoinductive material of claim 33, whereinthe mineralized particulate material is mineral recovered from bone. 35.The osteoinductive material of claim 33, wherein the mineralizedparticulate material is surface demineralized bone.
 36. Theosteoinductive material of claim 33, wherein the osteoinductive growthfactors are extracted from DBM.
 37. The osteoinductive material of claim33, wherein the osteoinductive growth factors are recovered fromdemineralization acid bath.
 38. An osteoinductive composition withreduced immunogenicity comprising: noncollagenous proteins extractedfrom demineralized bone or recovered from acid used to demineralizebone; and a carrier; wherein the osteoinductive composition exhibits anability to induce formation of heterotopic bone in a euthymic mouse. 39.The osteoinductive composition of claim 38, wherein the carrier ismineral recovered acid used to demineralize bone.
 40. The osteoinductivecomposition of claim 38, wherein the carrier is a demineralized bonematrix scaffold.
 41. An osteoinductive composition comprising: acarrier; and a protein composition comprising a protein recovered fromacid used to demineralize bone wherein the protein composition has lessthan 90% by weight inorganic components.
 42. The osteoinductivecomposition of claim 41, wherein the carrier is demineralized bone. 43.The osteoinductive composition of claim 41, wherein the carrier ishydroxyapatite.